LEAD EXPOSURE, HOMOCYSTEINE, DNA METHYLATION AND LATE-ONSET ALZHEIMER'S ...

198 Pages·2012·7.66 MB·English

LEAD EXPOSURE, HOMOCYSTEINE, DNA METHYLATION AND
LATE-ONSET ALZHEIMER’S DISEASE

by
Kelly M. Bakulski

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Environmental Health Sciences)
in The University of Michigan
2012

Doctoral Committee:

Professor Howard Hu, Co-Chair
Assistant Professor Laura S. Rozek, Co-Chair
Professor Henry L. Paulson
Assistant Professor Sung Kyun Park
© Kelly M. Bakulski
2012
ACKNOWLEDGMENTS

This dissertation was written by Kelly M. Bakulski, with extensive
contributions by Drs. Howard Hu, Dana Dolinoy, Laura Rozek, Henry Paulson,
Sung Kyun Park, Maureen Sartor, Bhramar Mukherjee, and Jennifer Weuve. I
would like to thank my dissertation committee members, Howard, Laura, Hank,
and Sung Kyun for the extensive time and mentorship they provided me. I would
also like to acknowledge Dr. Mukherjee for serving on my preliminary committee
and pointing me in the right direction from the start. Dana and Maureen also
provided extremely valuable guidance and mentorship. I feel very lucky to have
had this wide net of professional support.
I had the wonderful opportunity to have worked in the collaborative Hu,
Dolinoy, and Rozek laboratories and I am very grateful to my many, many
labmates for their assistance and advice. My Alzheimer’s project was supported
by the folks at the Michigan Alzheimer’s Research Center, including Hank, Dr.
Sid Gilman, Dr. Andrew Lieberman, Dr. Roger Albin, Dr. Bruno Giordani, Arijit
Bhaumik, Sherry Teboe, and Lisa Bain. The Normative Aging Study project was
mentored by Jennifer, Sung Kyun, and Dr. Kathleen Tucker.
I would like to extend special thanks to my family for their support, in
particular my mom, Barbara, my dad, David, my sister, Susan, and my
grandmother, Irene. I would also like to acknowledge the encouragement from
my Pauline roommates through the years, including Justin, Ashley, Murph,
Brenna, Heather, Lauren, Jon, and Luis.
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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
LIST OF FIGURES v
LIST OF TABLES vii
LIST OF ABBREVIATIONS ix
ABSTRACT x

CHAPTER
I. Introduction: Alzheimer’s disease and environmental exposure to 1
lead: The epidemiologic evidence and potential role of epigenetics
Abstract 1
Alzheimer’s disease 2
General Alzheimer’s disease epidemiology 2
Lead (Pb) exposure 5
Overview of Pb exposure as a risk factor 5
Pb exposure epidemiology 5
Biomarkers of Pb exposure 8
Pb as a neurotoxicant and risk factor for late-onset AD 8
Overview of epigenetics 10
Epigenetic epidemiology and Alzheimer’s disease 10
Epigenetics and heavy metals, with a focus on Pb 13
Data integrating Alzheimer’s disease, epigenetics and Pb 14
exposure
Alzheimer’s disease and Pb exposure are associated with 14
changes in one-carbon metabolism, the substrate for
DNA methylation
Animal model studies linking Pb exposure, epigenetics, and 16
amyloidogenesis
Challenges to LOAD epidemiologic research integrating 17
epigenetics and Pb exposure
Potential approaches to study Pb exposure, epigenomics, and 18
Alzheimer’s disease epidemiology
Figures 21
References 22
II. Research Chapter 1: Lead exposure, B-vitamins, and plasma 39
homocysteine in the VA Normative Aging Study
Abstract 39
Introduction 41
Methods 45
Results 49
iii
Discussion 52
Tables 57
Figures 64
References 68
Supplemental Tables 72
Supplemental Figures 76
III. Research Chapter 2: Genome-wide DNA methylation differences 77
between late-onset Alzheimer’s disease and cognitively normal
controls in the human frontal cortex.
Abstract 77
Introduction 79
Methods 81
Results 85
Discussion 91
Figures 97
Tables 104
References 108
IV. Research Chapter 3: An integrated analysis of genome-wide RNA 115
expression and DNA methylation in late onset Alzheimer’s
disease and neuropathological controls.
Abstract 115
Introduction 116
Results 119
Discussion 124
Methods 127
Figures 132
Tables 138
References 143
Supplemental Figures 148
Supplemental Tables 151
V. Conclusions and future directions 171
Overall summary 171
Summary of homocysteine (Hcy) and Pb exposure 172
Summary of DNA methylation in AD. 174
Summary of gene expression and DNA methylation in AD. 176
Future directions 177
Concluding remarks 181
References 182

iv
LIST OF FIGURES

Chapter 1
1.1 Conceptual diagram describing the relationship between 21
environmental exposures, including to the heavy metal lead, with
the development of late-onset Alzheimer’s disease.
Chapter 2
2.1 One-Carbon Metabolism Pathway. Homocysteine can be 64
elevated in conditions of low folate, low B6, or low B12. The
sulfhydryl groups on several proteins, including Cystathionine β-
synthase (CBS), in the one-carbon metabolism pathway are
potential sites for lead’s interferences.
2.2 The adjusted cross-sectional association between Pb exposure 65
and homocysteine.
2.3 Longitudinal core model. Tibia Pb. Data has been centered on 66
the mean continuous variables so intercept can be interpretable.
2.4 Adjusted association between Pb and homocysteine stratified by 67
nutrient status.
S 2.1 Bivariate scatterplots for the unadjusted associations between 76
lead measurement and Hcy at visit 1.
S 2.2 Blood Pb restricted plasma and diet. *Restrict analysis to only 76
individuals with both plasma and dietary measures.
Chapter 3
3.1 Mean percent methylation frequency distribution of the Discovery 97
Set of 12 cognitively normal control samples (A) and 12
Alzheimer’s disease cases (B) across the 27,578 CpG sites on
the Illumina HumanMethylation27 BeadArray.
3.2 Hierarchical clustering heatmap of the Discovery Set top 26 98
autosomal CpG loci associated with late-onset Alzheimer’s
disease (LOAD) case/control status after adjusting for sex and
age.
3.3 Discovery Set gene set enrichment analysis plots. 99
3.4 Human chromosome ideogram. 100
3.5 Methylation upstream of the TMEM59 gene. 101
3.6 Functional validation of observed DNA methylation differences for 102
TMEM59, a gene involved in the post-translational modification of
Amyloid Precursor Protein (AΒPP).
Chapter 4
v
4.1 Boxplots of gene expression of the 9 top genes that differ by 132
LOAD case or control status. LOAD cases display reduced gene
expression at all of 9 of the genes.
4.2 Heatmap of all of the 176 sites on the Affymetrix gene expression 133
array (FDR < 0.1) associated with LOAD case status. This plot
used maximum distance and Ward’s hierarchical clustering
methods. Data has been normalized to the mean expression
value per probeset.
4.3 Principal Component Analysis. (A) Principal component loading 134
histogram. (B). Principal component analysis scree plot for the top
176 probesets (FDR < 0.1).
4.4 Gene-expression and DNA methylation linked data analysis 135
pipeline.
4.5 (A) Scatterplot of the 133 genes that displayed discordant gene 135
expression and DNA methylation between LOAD cases and
controls.
4.6 Scatter plots of 9 genes: expression vs. methylation. 136
4.7 Circos plot: Locations of gene expression change and DNA 137
methylation change.
S 4.1 Heatmap of all of the 176 sites on the Affymetrix gene expression 148
array (FDR < 0.1) associated with LOAD case status. Publicly
available data from embryonic stem cell derived neuronal
precursor cells (GSE7178) and astrocytes (GSE5080) have been
included. This plot used maximum distance and Ward’s
hierarchical clustering methods and normalized to the mean
expression value per probeset.
S 4.2 Scatterplots of expression vs. methylation for the 23 genes 149
significantly associated with AD via expression (p<0.05) and
methylation (p<0.05), and methylation and expression are
significantly correlated (Pearson’s p<0.05).

vi
LIST OF TABLES

Chapter 2
2.1 Univariate Statistics: Characteristics of individuals with complete
Hcy and blood Pb (2234 obs, 1048 individuals). Mean (S.D.), 57
except where noted.
2.2 Bivariate statistics based on visit 1. Characteristics of individuals 5
with complete Hcy and blood Pb (1056 individuals). 58
2.3 Concurrent Pb exposure is associated with plasma 6
homocysteine: Mixed effects model, random intercept only. 60
Equivalent to cross-sectional model taking into account
correlated nature of observations from the same individual.
2.4 Cumulative exposure is associated with plasma homocysteine: 6
Mixed effects model, random intercept only. Equivalent to cross- 61
sectional model taking into account correlated nature of
observations from the same individual.
2.5 Longitudinal mixed effects models: Log(hcy) is outcome and tibia 6
pb is main predictor. Continuous covariates have been mean 62
adjusted so that the intercept can be interpreted as the log(hcy)
at the mean of those covariates and when dummy variables =0.
Four visits of tibia Pb and Hcy have been used. Random
intercept and slope.
2.6 Basic mediation analysis. All visits main effect of tibia Pb with 6
and without current blood Pb adjustment. Note: Tibia Pb is no 63
longer significant after adjusting for blood Pb
S 2.1 Visits 1-2 all samples, linear regression, split by change in blood 7
Pb n=747 72
S 2.2 All visits, all samples, mixed effects n=747 with 1245 7
observations 73
S 2.3 Secondary analysis, long-term dietary trend in tibia model 7
74
S 2.4 Mediation analysis, visit 1. 7
75
Chapter 3
3.1 Study population mean demographics by case status. 1
104
3.2 Pyrosequencing assay information. 1
105
3.3 Primer sequences for gene expression QPCR assays. 1
105
vii
3.4 CpG sites differentially methylated with age among cognitively 1
normal controls (Discovery Set, Age 69-94). 106
3.5 Table of the 25 CpG sites most significantly differentially 1
methylated by AD case status (Discovery Set). 107
Chapter 4
4.1 Affymetrix expression differences between LOAD cases and 1
controls (n=25) after adjusting for age and sex. 138
4.2 Among genes with lower gene expression in AD cases vs. 1
controls, the following are the top 15 biological processes that 139
are down-regulated based on LR-Path.
Among genes with higher gene expression in AD cases vs. 1
controls, the following are the top 15 biological processes that 140
are up-regulated based on LR-Path.
4.4 Gene expression and methylation correlation. 1
141
4.5 Study population characteristics. 1
142
S 4.1 Gene expression probesets associated with AD case vs. controls 1
at FDR <0.1 (n=176) 151
S 4.2 Among genes with lower expression in Alzheimer’s, several 1
biological processes were enriched. 156
S 4.3 Among genes with higher expression in Alzheimer’s, several 1
biological processes were enriched. 158
S 4.4 Among genes with lower expression in Alzheimer’s, several 1
molecular functions were enriched. 166
S 4.5 Among genes with higher expression in Alzheimer’s, several 1
molecular functions were enriched. 167
S 4.6 Among genes with lower expression in Alzheimer’s, the binding 1
motifs of several transcription factors were enriched. 169
S 4.7 Among genes with higher expression in Alzheimer’s, the binding 1
motifs of several transcription factors were enriched. 169
S 4.8 Among genes with higher expression in Alzheimer’s, the binding 1
sites of several microRNAs were enriched. 169
S 4.9 Among genes with lower expression in Alzheimer’s, the binding 1
sites of several microRNAs were enriched. 169
S 4.10 Among genes with higher expression in Alzheimer’s, several 1
cytogenic bands were enriched. 170
S 4.11 Among genes with lower expression in Alzheimer’s, several 1
cytogenic bands were enriched. 170

viii
LIST OF ABBREVIATIONS

8-oxo-dG 8-hydroxy-2'-deoxyguanosine
AD Alzheimer's disease
ADRC Alzheimer's disease research center
DOHad Developmental origins of adult health and disease
EOAD Early-onset Alzheimer's disease
EGCG Epigallocatechin gallate
GAWS Genome wide association study
HCY Homocysteine
KXRF Cd109 K-shell X-ray Fluorescence
LOAD Late-onset Alzheimer's disease
Pb Lead
MCI Mild cognitive impairment
NGS Next generation sequencing
PND Post natal day
SAM S-adenosyl methionine
SNP Single nucleotide polymorphism
NAS Veteran's Affairs Normative Aging Study
WBC White blood cell

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