A Novel Approach to Identify Candidate Imprinted Genes in ......Many imprinted genes are necessary...

A Novel Approach to Identify Candidate Imprinted Genes in Humans

by

Jonathan Samuel Shapiro

A thesis submitted in conformity with the requirements for the degree of Master of Science

Institute of Medical Science University of Toronto

© Copyright by Jonathan Samuel Shapiro 2012

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A Novel Approach to Identify Candidate Imprinted Genes in

Humans

Jonathan Samuel Shapiro

Master of Science

Institute of Medical Science University of Toronto

2012

Abstract

Many imprinted genes are necessary for normal human development. Approximately 70

imprinted genes have been identified in humans. I developed a novel approach to identify

candidate imprinted genes in humans using the premise that imprinted genes are often associated

with nearby parent-of-origin-specific DNA differentially methylated regions (DMRs). I

identified parent-of-origin-specific DMRs using sodium bisulfite-based DNA (CpG) methylation

profiling of uniparental tissues, mature cystic ovarian teratoma (MCT) and androgenetic

complete hydatidiform mole (AnCHM), and biparental tissues, blood and placenta. In support of

this approach, the CpG methylation profiling led to the identification of parent-of-origin-specific

differentially methylated CpG sites (DMCpGs) in known parent-of-origin-specific DMRs. I

found new DMRs for known imprinted genes NAP1L5 and ZNF597. Most importantly, I

discovered many new DMCpGs, which were associated with nearby genes, i.e., candidate

imprinted genes. Allelic expression analyses of one candidate imprinted gene, AXL, suggested

polymorphic imprinting of AXL in human blood.

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Acknowledgments

I would like to acknowledge my supervisor, Dr. Rosanna Weksberg, for her guidance,

encouragement, and patience. I would also like to acknowledge my program advisory committee

members Dr. Joseph Beyene, Dr. Andrew Paterson, and Dr. Sue Varmuza for their guidance and

support.

I would like to acknowledge the members of Dr. Rosanna Weksberg's laboratory. I would

particularly like to acknowledge Dr. Sanaa Choufani, Dr. Darci T. Butcher, Dr. Daria

Grafodatskaya, and Dr. Jose C. Ferreira for their guidance and support. I would also like to

acknowledge Chunhua Zhao, Youliang Lou, Sarah Goodman, Yi-An Chen, Kai-wei Chang, and

Khadine Wiltshire for their assistance.

I would like to acknowledge my collaborators Dr. Dalila Pinto and Dr. Stephen W. Scherer for

their assistance in my allelic expression analyses of AXL in humans and for producing the

idiogram showing the genomic regions of known and candidate parent-of-origin-specific

differentially methylated CpG sites (DMCpGs) in humans for our Genome Research journal

article.

I would also like to acknowledge Cold Spring Harbor Laboratory Press for publishing my work

in their journal Genome Research.

I would also like to acknowledge the Canadian Institutes of Health Research for the Frederick

Banting and Charles Best Canada Graduate Scholarships - Master's Award.

I would also like to acknowledge my family and friends for their support.

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Table of Contents

Acknowledgments ........................................................................................................................................................iii

Table of Contents .........................................................................................................................................................iv

List of Tables...............................................................................................................................................................vii

List of Figures ............................................................................................................................................................viii

List of Appendices........................................................................................................................................................ix

List of Abbreviations.....................................................................................................................................................x

Chapter 1 Introduction...................................................................................................................................................1

1 Introduction..............................................................................................................................................................1

1.1 Introduction to Imprinted Genes .....................................................................................................................1

1.1.1 Function and Expression Pattern of Imprinted Genes.......................................................................1

1.1.2 Discovery of Imprinted Genes ..........................................................................................................4

1.2 Uniparental Tissues - Androgenetic Complete Hydatidiform Mole (AnCHM) and Mature Cystic Ovarian Teratoma (MCT) .............................................................................................................................................8

1.3 Introduction to Epigenetics .............................................................................................................................9

1.3.1 DNA Methylation .............................................................................................................................9

1.3.2 Covalent Post-translational Modification of Histone Tails .............................................................18

1.3.3 Long Non-coding RNAs (ncRNAs)................................................................................................23

1.3.4 MicroRNAs (miRNAs)...................................................................................................................23

1.3.5 Crosstalk Between Epigenetic Mechanisms ...................................................................................23

1.4 Human Disorders Associated With Uniparental Origin Of Limited Genomic Regions................................25

1.4.1 Beckwith-Wiedemann syndrome (BWS)........................................................................................25

1.4.2 Russell-Silver syndrome (RSS) ......................................................................................................25

1.4.3 Prader-Willi syndrome (PWS) ........................................................................................................26

1.4.4 Angelman syndrome (AS) ..............................................................................................................26

1.5 Previous Methods Used to Find Candidate Imprinted Genes and Candidate Parent-of-Origin-Specific Differentially Methylated Regions (DMRs) in Humans ...............................................................................27

1.5.1 Searching for Candidate Parent-of-Origin-Specific DMRs Using Uniparental Tissues (AnCHMs and MCTs) ......................................................................................................................................27

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1.5.2 Searching for Candidate Imprinted Genes Using DNA Sequence Features Around Known Imprinted Genes..............................................................................................................................28

1.5.3 Searching for Candidate Imprinted Genes by Searching for Genes with Differential Allelic Expression.......................................................................................................................................29

1.5.4 Searching for Candidate Imprinted Genes by Searching for RNA Polymerase Bound to Only One of Two Alleles ................................................................................................................................29

1.5.5 Searching for Candidate Parent-of-Origin-Specific DMRs Using Tissue with Cells Containing Maternal/Paternal Uniparental Disomies for Human Chromosome 15...........................................30

1.5.6 Searching for Candidate Parent-of-Origin-Specific DMRs Using Biparental Tissues and Tissues with Cells Containing Genome-Wide Maternal/Paternal Uniparental Disomies ............................30

1.5.7 Searching for Candidate Parent-of-Origin-Specific DMRs Using Biparental Diploid Placentas, Diandric Triploid Placentas, Digynic Triploid Placentas, and Androgenetic Complete Hydatidiform Moles (AnCHMs).....................................................................................................31

1.6 Hypothesis and Aims ....................................................................................................................................33

Chapter 2 Materials and Methods................................................................................................................................35

2 Materials and Methods...........................................................................................................................................35

2.1 Sample Collection .........................................................................................................................................35

2.2 Sodium Bisulfite Treatment of DNA ............................................................................................................35

2.3 Microarray Processing...................................................................................................................................38

2.4 CpG Methylated Proportion and Detection P-value For Each Targeted CpG Site........................................39

2.5 Statistical Analyses to Characterize CpG Methylation .................................................................................39

2.6 Compiled List of Microarray CpG Sites in Known Parent-of-Origin-Specific DMRs Associated With Known Imprinted Genes ...............................................................................................................................42

2.7 Selection Criteria for Candidate Maternally Methylated CpG Sites in Blood...............................................47

2.8 Selection Criteria for Candidate Maternally Methylated CpG Sites in Placenta...........................................48

2.9 Selection Criteria for Candidate Paternally Methylated CpG Sites in Blood................................................49

2.9.1 Adapted Selection Criteria for Candidate Paternally Methylated CpG Sites in Blood ...................50

2.10 Selection Criteria for Candidate Paternally Methylated CpG Sites in Placenta ............................................52

2.10.1 Adapted Selection Criteria for Candidate Paternally Methylated CpG Sites in Placenta ...............53

2.11 Targeted Quantitative Sodium Bisulfite Pyrosequencing..............................................................................56

2.12 Box-and-whisker Plots ..................................................................................................................................58

2.13 Sodium Bisulfite Cloning/Sequencing ..........................................................................................................58

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2.14 Allelic Expression Analyses of AXL in Humans...........................................................................................59

Chapter 3 Results.........................................................................................................................................................63

3 Results ...................................................................................................................................................................63

3.1 CpG Methylation in Known Parent-of-Origin-Specific DMRs Associated with Known Imprinted Genes..63

3.2 Candidate Parent-of-Origin-Specific Differentially Methylated CpG Sites (DMCpGs)...............................64

3.3 Allelic Expression of AXL in Humans...........................................................................................................67

Chapter 4 Discussion...................................................................................................................................................85

4 Discussion..............................................................................................................................................................85

4.1 More Complete Picture of CpG Methylation in Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes) ..............................................................................................................................85

4.2 Biparental Tissues (When Compared to Uniparental Tissues) Have Differential CpG Methylation in Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes) .............................................86

4.3 Variable CpG Methylation in Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes) ...........................................................................................................................................................87

4.4 Poor Microarray Coverage of Known Paternally Methylated DMRs ...........................................................88

4.5 Very Few Candidate Imprinted Loci Identified By More Than One Study ..................................................89

4.6 Boundaries of Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes) are Unknown.......................................................................................................................................................91

4.7 Candidate Maternally Methylated DMR in Promoter of Imprinted Gene NAP1L5 ......................................92

4.8 Candidate Paternally Methylated DMR in Promoter of Imprinted Gene ZNF597 ........................................92

4.9 Candidate Maternally Methylated DMR in Promoter of RIMS2...................................................................93

4.10 Candidate Paternally Methylated DMR in Promoter of AXL ........................................................................93

4.11 Polymorphic Imprinting of AXL....................................................................................................................93

4.12 General Discussion........................................................................................................................................94

4.13 Future Directions...........................................................................................................................................95

4.14 Conclusions ...................................................................................................................................................96

References ...................................................................................................................................................................98

Appendices ................................................................................................................................................................128

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List of Tables

Table 3-1: AXL SNP Quantification in DNA and RNA of Informative Individuals 83

Table A-1: DNA Methylation in Known Parent-of-Origin-Specific DMRs 129

Table A-2: Description of Blood Samples 140

Table A-3: Description of Placenta Samples 141

Table A-4: Number of CpG Methylated Proportions Replaced with “N/A” 142

Table A-5: Array CpG Sites Located in DMRs Associated with Known Imprinted Genes 143

Table A-6: Candidate Maternally Methylated CpG Sites in Blood 144

Table A-7: Candidate Maternally Methylated CpG Sites in Placenta 147

Table A-8: Candidate Paternally Methylated CpG Sites in Blood 151

Table A-9: Candidate Paternally Methylated CpG Sites in Placenta 152

Table A-10: PCR Conditions for Bisulfite Pyrosequencing 153

Table A-11: PCR Cycling Conditions for Bisulfite Pyrosequencing 154

Table A-12: Primer Sequences for PCR for Bisulfite Pyrosequencing 155

Table A-13: Primer Sequences For Bisulfite Pyrosequencing Reactions 156

Table A-14: PCR Conditions for Sodium Bisulfite Sequencing 157

Table A-15: PCR Cycling Conditions for Sodium Bisulfite Sequencing 158

Table A-16: Primer Sequences for PCR for Sodium Bisulfite Sequencing 159

Table A-17: PCR Conditions for SNP (rs1051008) Pyrosequencing 160

Table A-18: PCR Cycling Conditions for SNP (rs1051008) Pyrosequencing 161

Table A-19: Primer Sequences for PCR for SNP (rs1051008) Pyrosequencing 162

Table A-20: Primer Sequence for SNP (rs1051008) Pyrosequencing Reactions 163

Table A-21: Overlapping Candidate Maternally Methylated CpG sites 164

Table A-22: Overlapping Candidate Paternally Methylated CpG sites 165

Table A-23: Candidate DMCpGs That May Represent Components of Known DMRs 166

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List of Figures

Figure 1-1: Locations of Known Imprinted Regions in the Human and Mouse Genomes 2

Figure 1-2: Imprinted Regions/Domains on Human Chromosome 11p15 16

Figure 1-3: Chromatin: DNA and its Associated Proteins 19

Figure 1-4: Location of Covalent Post-translational Histone Tail Modifications 21

Figure 2-1: Steps in the Sodium Bisulfite-Mediated Deamination of Cytosine 36

Figure 2-2: Distribution of Unadjusted and FDR-adjusted Mann-Whitney P-values 43

Figure 2-3: Expected CpG Methylation in Maternally/Paternally Methylated DMRs 45

Figure 3-1: CpG Methylation in DMRs Associated With Known Imprinted Genes 68

Figure 3-2: CpG Methylation within Four DMRs Associated with Imprinted Genes 70

Figure 3-3: CpG Methylation within the Candidate NAP1L5 DMR 73

Figure 3-4: CpG Methylation within the Candidate ZNF597 DMR 75

Figure 3-5: CpG Methylation within the Candidate RIMS2 DMR 77

Figure 3-6: CpG Methylation within the Candidate AXL DMR 79

Figure 3-7: Candidate AXL DMR Sodium Bisulfite Sequencing 81

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List of Appendices

Table A-1: DNA Methylation in Known Parent-of-Origin-Specific DMRs 129

Table A-2: Description of Blood Samples 140

Table A-3: Description of Placenta Samples 141

Table A-4: Number of CpG Methylated Proportions Replaced with “N/A” 142

Table A-5: Array CpG Sites Located in DMRs Associated with Known Imprinted Genes 143

Table A-6: Candidate Maternally Methylated CpG Sites in Blood 144

Table A-7: Candidate Maternally Methylated CpG Sites in Placenta 147

Table A-8: Candidate Paternally Methylated CpG Sites in Blood 151

Table A-9: Candidate Paternally Methylated CpG Sites in Placenta 152

Table A-10: PCR Conditions for Bisulfite Pyrosequencing 153

Table A-11: PCR Cycling Conditions for Bisulfite Pyrosequencing 154

Table A-12: Primer Sequences for PCR for Bisulfite Pyrosequencing 155

Table A-13: Primer Sequences For Bisulfite Pyrosequencing Reactions 156

Table A-14: PCR Conditions for Sodium Bisulfite Sequencing 157

Table A-15: PCR Cycling Conditions for Sodium Bisulfite Sequencing 158

Table A-16: Primer Sequences for PCR for Sodium Bisulfite Sequencing 159

Table A-17: PCR Conditions for SNP (rs1051008) Pyrosequencing 160

Table A-18: PCR Cycling Conditions for SNP (rs1051008) Pyrosequencing 161

Table A-19: Primer Sequences for PCR for SNP (rs1051008) Pyrosequencing 162

Table A-20: Primer Sequence for SNP (rs1051008) Pyrosequencing Reactions 163

Table A-21: Overlapping Candidate Maternally Methylated CpG sites 164

Table A-22: Overlapping Candidate Paternally Methylated CpG sites 165

Table A-23: Candidate DMCpGs That May Represent Components of Known DMRs 166

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List of Abbreviations

AnCHM androgenetic complete hydatidiform mole

AP-2 activating protein 2

AS Angelman syndrome

AXL anexelekto

BWS Beckwith-Wiedemann syndrome

cDNA copy deoxyribonucleic acid

CGIs CpG islands

CML chronic myeloid/myelogenous leukemia

CMLs chronic myeloid/myelogenous leukemias

CpG cytosine connected to a guanine through a phosphate group

CRC colorectal cancer

CRCs colorectal cancers

CTCF CCCTC-binding factor

DMCpG differentially methylated CpG site

DMCpGs differentially methylated CpG sites

DMR differentially methylated region

DMRs differentially methylated regions

DNA deoxyribonucleic acid

DNMT DNA methyltransferase

DNMTs DNA methyltransferases

dpc days post coitus

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EC esophageal cancer

ECs esophageal cancers

EMT epithelial to mesenchymal transition

FDR false discovery rate

GOM gain of methylation

H2 histone 2

H3 histone 3

H3K27 histone 3 lysine 27



H4 histone 4


HDAC histone deacetylase

hESC human embryonic stem cell

HP1 heterochromatin protein 1

IC1 imprinting centre 1

IC2 imprinting centre 2

ICC intraclass correlation coefficient

ID identifier

IQR interquartile range

KMMDMRs known maternally methylated DMRs associated with known imprinted genes

KPMDMRs known paternally methylated DMRs associated with known imprinted genes

LCL lymphoblastoid cell line

xii

LCLs lymphoblastoid cell lines

LOI loss of imprinting

LOM loss of methylation

MAT maternally inherited chromosome

MBD methyl-CpG binding domain

MCT mature cystic ovarian teratoma

MeCP methyl-cytosine binding protein

MeDIP methylated DNA immunoprecipitation

miRNA microRNA

mUPD maternal uniparental disomy

mUPD11 maternal uniparental disomy for chromosome 11

mUPD14 maternal uniparental disomy for chromosome 14

N number of values

N/A Not Applicable

ncRNA non-coding RNA

ncRNAs non-coding RNAs

PAT paternally inherited chromosome

PCR Polymerase Chain Reaction

PRC1 polycomb repressive complex 1

pUPD paternal uniparental disomy

pUPD11 paternal uniparental disomy for chromosome 11

pUPD11p15 paternal uniparental disomy for chromosome region 11p15


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PWS Prader-Willi syndrome

RCC renal cell carcinoma

RCCs renal cell carcinomas

RNA ribonucleic acid

RNAs ribonucleic acids

ROI retention of imprinting

RSS Russell-Silver syndrome

snoRNA small nucleolar RNA

SNP single-nucleotide polymorphism

SP1 specificity protein 1

SP3 specificity protein 3

Std. Dev. standard deviation

TCAG The Centre for Applied Genomics

tDMR tissue-specific differentially methylated region

UPD uniparental disomy

UPDs uniparental disomies

UPD11p15 uniparental disomy for chromosome 11 region 11p15

UPD14 uniparental disomy for chromosome 14

UPD4 uniparental disomy for chromosome 4

WBC white blood cell

WGA whole genome amplification

YY1 Ying Yang 1

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Chapter 1 Introduction

1 Introduction

1.1 Introduction to Imprinted Genes

Imprinted genes are expressed or silenced depending on their parent-of-origin, either the

maternal or paternal chromosome. This parent-of-origin-specific expression may be tissue-

specific and/or developmental-stage-specific1-3. There are approximately 70 known imprinted

genes in humans and 100 known imprinted genes in mice4. These known imprinted genes are

located in imprinted regions that map across the genome (Figure 1-1). Although there are many

different mechanisms that regulate the expression pattern of imprinted genes5-12, imprinted gene

expression patterns are usually associated with parent-of-origin-specific DNA differentially

methylated regions (DMRs)5,7,8,13-29 (discussed later in “DNA Methylation and Imprinted Genes”

section).

1.1.1 Function and Expression Pattern of Imprinted Genes

Imprinted genes have been implicated in many different biological processes30-67, including

development. Imprinted genes Igf2 and Ube3a have been implicated in development 46,47. In

murine conceptuses, reduced expression of functional Igf2 (achieved by knocking out the

preferentially expressed allele) leads to fetal and placental growth restriction46, whereas

overexpression of functional Igf2 (achieved by generating chimeric conceptuses by inserting

embryonic stem cells expressing functional transgenic Igf2 into conceptuses at the blastocyst

stage) leads to fetal overgrowth68. Reduced expression of functional Ube3a (achieved by

knocking out the maternal copy of Ube3a) leads to motor dysfunction in mice47.

Imprinted genes Igf2 and Ube3a demonstrate the complex regulation of imprinted genes. In

mice, Ube3a is usually paternally and maternally expressed in most tissues, while Ube3a is

usually paternally silent and maternally expressed in brain2. Igf2 is usually paternally expressed

and maternally silent in most mouse tissues, but expressed from both parental chromosomes in

mouse central nervous system neurons1.

2

Figure 1-1: Locations of Known Imprinted Regions in the Human and Mouse Genomes

Idiograms showing the genomic locations of known imprinted regions (i.e., regions with known imprinted genes) in

the human (A) and mouse (B) genomes. The red filled triangles point to genomic locations of known imprinted

regions. Some known imprinted regions contain only one known imprinted gene, whereas other known imprinted

regions contain 14+ known imprinted genes.

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A

B

Figure 1-1: Locations of Known Imprinted Regions in the Human and Mouse Genomes

4

1.1.2 Discovery of Imprinted Genes

Extensive research on the reasons why parthenogenesis, “the process by which an egg can

develop into an embryo in the absence of sperm”69, does not occur in mammals when it does in

other classes of vertebrates, such as insects and reptiles, led to the discovery of imprinted genes.

Kaufman and Sachs (1975)70 described the death of parthenogenetic embryos in mice. The

reasons for the deaths of mouse parthenogenetic embryos was not immediately known. With the

combined work of Surani et al. (1984)71 and McGrath and Solter (1984)72, it was shown that both

sperm and oocyte pronuclei were required for normal development in mouse conceptuses.

Surani et al (1984)71 used nuclear transplantation to transplant a donor oocyte pronucleus into a

recipient oocyte to create a diploid parthenogenetic (2 maternal genomes) conceptus. They also

used nuclear transplantation to transplant a donor sperm pronucleus into a recipient oocyte to

create a diploid biparental (1 maternal and 1 paternal genome) conceptus. The diploid biparental

conceptuses developed to term. However, the diploid parthenogenetic conceptuses did not

develop to term as they died during gestation and were absorbed into the endometrium.

McGrath and Solter (1984)72 used nuclear transplantation of pronuclei between zygotes to

generate diploid androgenetic (2 paternal genomes) and diploid gynogenetic (2 maternal

genomes) conceptuses (gynogenesis “is a form of development in which eggs are activated by

sperm which does not contribute genetically to the resulting embryo”73). They also used nuclear

transplantation of pronuclei between zygotes to generate diploid biparental (1 maternal and 1

paternal genome) conceptuses to compare to their diploid androgenetic and gynogenetic

conceptuses. The diploid biparental conceptuses developed to term, whereas the diploid

androgenetic and gynogenetic conceptuses did not. Both the diploid androgenetic and

gynogenetic conceptuses died during gestation and were absorbed into the endometrium. Diploid

gynogenetic conceptuses developed embryonic tissues, but very little to no trophoblastic tissue in

comparison to diploid biparental conceptuses. Diploid androgenetic conceptuses developed

trophoblastic tissue, but very little embryonic tissues in comparison to diploid biparental

conceptuses. Barton et al. (1984)74 suggested sperm pronuclei are necessary for the development

of extraembryonic tissues such as trophoblast, whereas oocyte pronuclei are necessary for some

phases of embryogenesis.

5

The existence of imprinted genes was also suggested by studies of mice with paternal and

maternal uniparental disomies (UPDs)75. The term paternal uniparental disomy (pUPD) refers to

cells that have two homologous chromosomes from the father instead of one homologous

chromosome from each parent. The term maternal uniparental disomy (mUPD) refers to cells

that have two homologous chromosomes from the mother instead of one homologous

chromosome from each parent. Paternal and maternal uniparental disomies (UPDs) for the same

genomic region had opposite effects on development. Mice with a maternal uniparental disomy

for chromosome 11 (mUPD11) were small, whereas mice with a paternal uniparental disomy for

chromosome 11 (pUPD11) were large, when compared to mice with biparental inheritance of

chromosome 1175. Mice with a maternal uniparental disomy for distal chromosome 2 were

hypoactive, whereas mice with a paternal uniparental disomy for distal chromosome 2 were

hyperactive, in comparison to mice with a biparental inheritance for distal chromosome 275.

1.1.2.1 Studies that Led to the Discovery of Mouse Igf2r Imprinting

In 1962, in the Jackson Laboratory (Bar Harbor, Maine), a mouse from an AKR/J strain was

born with a hair-pin tail76. The mutation, a deletion, that caused the hairpin-tail phenotype was

identified and referred to as Thp. Johnson (1974)76 studied the phenotypic effect of the hairpin

mutation (Thp). He noted that if a mouse embryo inherited the Thp mutation maternally (i.e., via

the egg), the mouse embryo typically died late in gestation. He also noted that if a mouse embryo

inherited the Thp mutation paternally (i.e., via the sperm), the mouse embryo was viable.

McGrath and Solter (1984)77 carried out reciprocal nuclear transplantation experiments between

one-cell embryos generated using sperm from +/+ males and oocytes from Thp /+ and +/+

females. They observed that the maternally inherited lethal effect was preserved when Thp /+

pronuclei were transplanted into +/+ one-cell embryos (that had their +/+ pronuclei removed).

They also observed that the maternally inherited lethal effect was not preserved when +/+

pronuclei were transplanted into Thp /+ one-cell embryos (that had their Thp /+ pronuclei

removed). These results suggested that the lethal effect was inherited via the oocyte’s pronuclei,

not the oocyte’s cytoplasm.

6

Winking and Silver (1984)78 suggested that deletion of a locus, which they termed the Tme (T-

associated maternal effect) locus, was responsible for the maternally inherited lethal effect. They

noted that the Tme locus is located within the genomic region deleted in the Thp mutation. They

also noted that the Tme locus is deleted in other chromosomal variations that lead to the same

maternally inherited lethal effect.

Barlow et al. (1991)79 performed expression analyses for genes located within the Tme locus

using mouse embryos that inherited Tme locus deletions from either parent, as well as mouse

embryos that did not inherit a Tme locus deletion. They observed expression of Igf2r in mouse

embryos that inherited the Tme locus deletion paternally (i.e., via the sperm). They also observed

expression of Igf2r in mouse embryos that did not inherit a Tme locus deletion. However, they

did not observe expression of Igf2r in mouse embryos that inherited the Tme locus deletion

maternally (i.e., via the egg). These results suggested Igf2r has parent-of-origin-specific

expression, which is characteristic of an imprinted gene. The other three genes (Tcp1, Plg, and

Sod2) located within the Tme locus they assayed had expression in all mouse embryos,

suggesting that these three genes do not have parent-of-origin-specific expression in mouse

embryos.

1.1.2.2 Studies that Led to the Discovery of Mouse Igf2 Imprinting

Searle and Beechey (1990)80 using crosses of mice with translocations involving chromosome 7

showed that a maternal duplication (or a paternal deficiency) of distal chromosome 7 results in

late prenatal lethality. They also demonstrated that a paternal duplication (or a maternal

deficiency) of distal chromosome 7 results in early embryonic lethality.

DeChiara et al. (1990)48 generated chimeric mice with a targeted disruption in the gene encoding

insulin-like growth factor II (IGF-II). They noted that if a mouse inherited the deletion

maternally (i.e., via the egg), the mouse developed normally. They noted, however, that if a

mouse inherited the deletion paternally (i.e., via the sperm), the mouse became growth deficient.

They also noted that mice homozygous for the deletion were phenotypically indistinguishable

from the heterozygous mice that inherited the deletion paternally. They performed expression

analyses for Igf2 using mouse embryos that inherited the Igf2 deletion from their father, as well

7

as mouse embryos that did not inherit the Igf2 deletion. They observed expression of Igf2 in all

mouse embryos. However, the expression of Igf2 in mouse embryos that inherited the Igf2

deletion from their father was 10-fold less when compared to mouse embryos that did not inherit

the Igf2 deletion.

DeChiara et al. (1991)1 performed nuclease protection assays on RNA from mouse embryos that

inherited the Igf2 deletion from either parent, as well as mouse embryos that did not inherit the

Igf2 deletion. They observed expression of Igf2 in mouse embryos and placentas that inherited

the Igf2 deletion maternally. They also observed expression of Igf2 in mouse embryos and

placentas that did not inherit the Igf2 deletion. However, they did not observe expression of Igf2

in mouse embryos and placentas that inherited the Igf2 deletion paternally. These results

suggested Igf2 has parent-of-origin-specific expression, which is a characteristic of imprinted

genes.

1.1.2.3 Studies that Led to the Discovery of Human H19 Imprinting

Bartolomei et al. (1991)81 performed nuclease protection assays on RNA from hybrid offspring

of four subspecies of Mus. They observed that only the maternally derived (i.e., via the egg) H19

gene was expressed. These results suggested mouse H19 has parent-of-origin-specific

expression, which is a characteristic of imprinted genes.

Zhang and Tycko (1992)82 performed H19 expression analyses (using informative transcribed

polymorphisms, restriction enzymes, and sequencing of PCR products) on genomic DNA and

cDNA (derived from RNA) from human fetal organs (liver, kidney, lung, heart, adrenal gland,

spleen, thymus, brain, leptomeninges, and placenta). They observed monoallelic expression of

H19 in all tissues (except placenta). They suggested the biallelic expression of H19 in placenta

may be due to maternal decidua contamination in the collected placenta samples.

Rachmilewitz et al. (1992)83 performed H19 expression analyses in human first trimester

placentas, term placentas, and androgenetic complete hydatidiform moles (discussed later in

“Uniparental Tissues” section). The H19 expression observed in androgenetic complete

hydatidiform moles was less than 10% the H19 expression observed in first trimester and term

placentas.

8

1.2 Uniparental Tissues - Androgenetic Complete Hydatidiform Mole (AnCHM) and Mature Cystic Ovarian Teratoma (MCT)

An androgenetic complete hydatidiform mole (AnCHM) is a conceptus with excessive

proliferation of villi and absent embryonic development84,85. AnCHMs occur naturally (i.e.,

without human intervention) in humans and are similar to diploid androgenetic mouse

conceptuses (mentioned previously in “Discovery of Imprinted Genes” section) in that they both

contain two paternal (i.e., sperm-derived) genomes and consist mainly of trophoblastic tissue72,86-

89. Kajii and Ohama (1977)86 suggested AnCHMs may result from the fertilization of an empty

(i.e., no maternal genome) egg by a haploid sperm that has its genome duplicated after

fertilization. Kajii and Ohama (1977)86 also suggested AnCHMs may arise from the fertilization

of an empty (i.e., no maternal genome) egg by two haploid sperms or by a diploid sperm.

A mature cystic ovarian teratoma (MCT) is a dermoid cyst containing differentiated tissues that

arise from embyronal ectoderm, mesoderm, and endoderm90. MCTs occur naturally (i.e., without

human intervention) in humans and are similar to diploid gynogenetic mouse conceptuses

(mentioned previously in “Discovery of Imprinted Genes” section) in that they both contain two

maternal (i.e., oocyte-derived) genomes and consist of differentiated tissues from all three

embryonal germ cell layers (i.e., ectoderm, mesoderm, and endoderm)91,92. Linder et al. (1975)93

suggested MCTs may result from a single female germ cell after the first meiotic division and the

subsequent failure of meiosis II. Other studies have suggested MCTs may arise from any stage of

female oogenesis94-96.

9

1.3 Introduction to Epigenetics

Epigenetics refers to “the study of any potentially stable and, ideally, heritable change in gene

expression or cellular phenotype that occurs without changes in” DNA nucleotide sequence 97.

Epigenetic mechanisms influence gene expression without changing the primary DNA sequence.

Epigenetic mechanisms, such as DNA methylation, covalent post-translational modification of

histone tails, and long non-coding RNAs (ncRNAs), regulate the expression patterns of

imprinted genes5,7,10-12. Epigenetic mechanisms play important roles in maintaining transposable

elements in their inactive state, inactivating X chromosomes, differentially marking parental

alleles of imprinted regions, and maintaining genome stability and integrity6,98-106.

1.3.1 DNA Methylation

DNA methylation is the epigenetic modification that has been most widely studied in mammals

with respect to normal development and disease states.

1.3.1.1 DNA methyltransferases (DNMTs)

In vertebrates, DNA becomes methylated on the carbon-5 position of the cytosine ring by DNA

methyltransferase enzymes, DNMT1, DNMT3a, and DNMT3b107-109.

DNMT1 methylates hemi-methylated CpG sites and during DNA replication it is recruited to

replication foci to faithfully replicate the CpG methylation state in the daughter strand110,111.

Mouse embryos with homozygous Dnmt1 deletions die at approximately 10-11 days post coitus

(dpc), demonstrating Dnmt1 is necessary for normal embryonic development112.

DNMT3a and DNMT3b methylate unmethylated CpG sites113. When a DNA methyltransferase

methylates an unmethylated CpG site, the DNA methylation is considered de novo. Mice with

homozygous Dnmt3a deletions die shortly (4 weeks) after birth, indicating Dnmt3a is essential

for ordinary development114. Mouse embryos with homozygous Dnmt3b deletions die before

birth, showing Dnmt3b is required for typical fetal development114.

10

DNMT3l shares sequence identity with DNMT3a and DNMT3b, but DNMT3l lacks the catalytic

domain of DNMT3a and DNMT3b115. DNMT3l interacts with DNMT3a and DNMT3b to

facilitate the interaction with targets to be methylated116. Mice with homozygous Dnmt3l

deletions are sterile117. Germ cells in male mice with homozygous Dnmt3l deletions fail to

mature and undergo apoptosis118. Oogenesis appears to be normal in female mice with

homozygous Dnmt3l deletions117. However, the offspring of female mice with homozygous

Dnmt3l deletions mated with male wild-type mice die at approximately 9.5 days post coitus

(dpc)117. Bourc’his et al. (2001)117 suggested Dnmt3l may play a role in establishing imprinted

gene expression patterns as Dnmt3l is usually only expressed in germ cells when differential

DNA methylation imprints on the maternal and paternal chromosomes are established.

1.3.1.2 CpG Islands (CGIs)

The majority of cytosine methylation occurs in CpG dinucleotides, cytosines that are followed by

guanines on the same strand of DNA119. CpG dinucleotides are underrepresented in vertebrate

genomes; occurring at 20% of their expected frequency120,121. CpG islands are regions of the

genome rich in cytosine, guanine, and CpG dinucleotides122. Gardiner-Garden and Frommer

(1987)122 defined CpG islands as genomic regions with cytosine-guanine content of 50% or

more, length greater than 200 base pairs, and a ratio greater than 0.6 of the observed number of

CpG dinucleotides to the expected number of CpG dinucleotides in the given genomic region. In

a later study, Takai and Jones (2002)123 noted definitions of CpG islands are subjective and

defined CpG islands as genomic regions with cytosine-guanine content of 55% or more, length

greater than 500 base pairs, and a ratio greater than 0.65 of the observed number of CpG

dinucleotides to the expected number of CpG dinucleotides in the given genomic region. CpG

islands occur in approximately 40% of mammalian gene promoters124. The conservation of these

CpG islands, as well as the high number of transcription factor binding sites containing CpG

sites in their consensus sequences demonstrate the importance of these regions125,126.

11

1.3.1.3 Gene Silencing

Cytosine methylation within transcription factor binding sites can deter transcription factor

binding, thereby affecting gene expression127-130. For example, transcription factors such as

activating protein 2 (AP-2), specificity protein 1 (SP1), specificity protein 3 (SP3), and Ying

Yang 1 (YY1) cannot bind to methylated cytosines in their transcription factor binding sites128-

130. If these transcription factors cannot bind, the recruitment of transcriptional machinery will be

affected, leading to a reduction in gene expression. For example, methylation at the transcription

factor YY1 DNA binding site in the promoter region of imprinted gene Peg3 leads to decreased

Peg3 expression130.

Cytosine methylation can also influence transcription by inhibiting the binding of transcriptional

regulators to their DNA binding sites. For example, the transcriptional regulator CCCTC-binding

factor (CTCF) has reduced binding affinity depending on the density and location of methylated

cytosines in its DNA binding site5,131. Human CTCF has eleven zinc finger DNA binding

domains132. CTCF binds to DNA using different combinations of its zinc finger DNA binding

domains132-136. The human genome contains roughly 15,000 CTCF binding sites137,138. CTCF has

been implicated in the activation and repression of many genes5,132,134-136. For example, CTCF

binding in imprinting centre 1 (IC1) on maternal human chromosome 11p15.5 is associated with

increased expression of the maternally expressed imprinted gene H19 and decreased expression

of the paternally expressed imprinted gene IGF29.

Aberrant DNA methylation patterns have been observed in cancer. Whereas cancers generally

demonstrate genome-wide hypomethylation, hypermethylation at the promoters of specific genes

plays an important role in tumourigenesis139. DNA methylation silences tumour-suppressor and

DNA damage repair genes, leading to a predisposition to cancer139-154. DNA hypermethylation

has been observed in the promoter region of BRCA1, a tumour suppressor gene, in breast and

ovarian cancer when compared to non-cancerous breast and ovarian tissue140,155,156. DNA

methylation in the promoter region of BRCA1 silences BRCA1157-159. DNA hypermethylation

has also been observed in the promoter region of MLH1, a gene involved in DNA damage repair,

in sporadic colorectal cancer when compared to non-cancerous colon tissue160. DNA methylation

in the promoter region of MLH1 silences MLH1161.

12

1.3.1.4 DNA Methylation and Imprinted Genes

The parent-of-origin-specific expression pattern of imprinted genes has been demonstrated in

association with parent-of-origin-specific differentially methylated regions (DMRs). Many

studies have assessed cytosine methylation within these regions (Appendices Table A-

1)16,18,21,24,28,162-205. Aberrant cytosine methylation within some of these regions has been

associated with human disorders206-209 (discussed later in “Human Disorders Associated With

Uniparental Origin Of Limited Genomic Regions” section). Classic examples of parent-of-

origin-specific DMRs (associated with imprinted genes) are imprinting centre 1 (IC1) and

imprinting centre 2 (IC2) on human chromosome 11p15 (Figure 1-2)210.

1.3.1.4.1 Imprinting Centre 1 (IC1)

IC1, also known as H19DMR, is a primary parent-of-origin-specific DMR, a parent-of-origin-

specific DMR whose parent-of-origin-specific CpG methylation is inherited from the germline,

located between maternally expressed imprinted gene H19 and paternally expressed imprinted

gene IGF2. IC1 is usually methylated in sperm and unmethylated in ova173,211. The developing

embryo and placenta inherit these parental methylation patterns. Since IC1 is usually methylated

on the paternally inherited chromosome and unmethylated on the maternally inherited

chromosome, IC1 is referred to as a paternally methylated DMR. In contrast, an imprinting

centre (IC) that is usually unmethylated on the paternally inherited chromosome and methylated

on the maternally inherited chromosome is referred to as a maternally methylated DMR.

Usually the maternal unmethylated IC1 is bound by CTCF, a transcriptional regulator, resulting

in expression of H19 and silencing of Igf25,131,212. The CTCF bound to unmethylated IC1 causes

interference in the competition between H19 and Igf2 for a set of common downstream

enhancers (3’ of H19). When CTCF is bound to unmethylated IC1, Igf2 no longer has access to

the H19 downstream enhancers. As a result, H19 is expressed and Igf2 is silenced. In contrast,

CTCF does not bind to the paternal methylated IC1. Paternal Igf2 has access to the H19

downstream enhancers, resulting in expression of Igf2 and silencing of H19.

Loss of imprinting (LOI) of IGF2, i.e., expression of IGF2 from both maternal and paternal

chromosome 11, has been observed in some cancers that have retention of imprinting (ROI) of

13

IGF2, i.e., IGF2 paternally expressed and maternally silent, in adjacent non-cancerous tissue.

Loss of imprinting (LOI) of IGF2 has been noted in 100% of advanced chronic

myeloid/myelogenous leukemias (CMLs)213, 40-70% of Wilm’s tumours (nephroblastomas)214-

217, 30-65% of colorectal cancers (CRCs)201,218-220, ~50% of renal cell carcinomas (RCCs)221,222,

and 20-55% of esophageal cancers (ECs)223,224. The general population’s lifetime risk of

colorectal cancer (CRC) and renal cell carcinoma (RCC) is approximately 1 in 20225 and 1 in

74226, respectively. From the above values, it can be roughly estimated that the general

population’s lifetime risk of colorectal cancer (CRC) with loss of imprinting (LOI) of IGF2 is

between 3 in 200 and 13 in 400. It can also be roughly estimated that the general population’s

lifetime risk of renal cell carcinoma (RCC) with loss of imprinting (LOI) of IGF2 is 1 in 148.

The loss of imprinting (LOI) of IGF2 in Wilm’s tumours (nephroblastomas) and colorectal

cancers (CRCs) was observed in association with a gain of methylation (GOM) on the normally

unmethylated IC1201,203. The loss of imprinting (LOI) of IGF2 in colorectal cancers (CRCs) was

also observed in association with a loss of methylation (LOM) on the normally methylated

maternally inherited copy of IGF2 DMR0 (the paternally inherited copy of IGF2 DMR0 is

usually unmethylated)227. IGF2 DMR0 is an example of a secondary parent-of-origin-specific

DMR, which is a parent-of-origin-specific DMR whose parent-of-origin-specific CpG

methylation is established after fertilization.

In mice, a targeted deletion of murine IC1 on paternal chromosome 7 did not interfere with the

imprinted gene expression pattern of Igf2; paternally expressed imprinted gene Igf2 was

expressed on the paternal chromosome 7 with the targeted deletion of IC1228. A targeted deletion

of murine IC1 on maternal chromosome 7 caused the normally silent paternally expressed

imprinted gene Igf2 to be expressed on the maternal chromosome 7 with the targeted deletion of

IC1; loss of imprinting (LOI) of Igf2 occurred in mice with a targeted deletion of murine IC1 on

maternal chromosome 7 and a wild-type paternal chromosome 7228. The expression of Igf2 in

mice with a targeted deletion of murine IC1 on maternal chromosome 7 and a wild-type paternal

chromosome 7 was approximately twice the expression of Igf2 in wild-type littermates228. For

comparison, the expression of IGF2 in Wilm’s tumours (nephroblastomas) with loss of

imprinting (LOI) of IGF2 was approximately double the expression of IGF2 in adjacent non-

cancerous colonic tissue with retention of imprinting (ROI) of IGF2229. Mice with a targeted

deletion of murine IC1 on maternal chromosome 7 and a wild-type paternal chromosome 7 were

14

heavier at birth than their wild-type littermates228. Mice with a targeted deletion of murine IC1

on maternal chromosome 7 and a wild-type paternal chromosome 7 developed approximately

double the number of adenomas as wild-type littermates230.

1.3.1.4.2 Imprinting Centre 2 (IC2)

Another classic example of a primary parent-of-origin-specific DMR associated with parent-of-

origin-specific gene expression is imprinting centre 2 (IC2), which is also known as KvDMR1.

This DMR is located within intron 9 of imprinted gene KCNQ1. IC2, associated with paternally

expressed imprinted gene KCNQ1OT1 and maternally expressed imprinted genes KCNQ1,

KCNQ1DN, CDKN1C, SLC22A18, and PHLDA28,9, is usually unmethylated in sperm and

methylated in ova169. CpG methylation at IC2 silences the expression of KCNQ1OT1, a long

non-coding RNA that starts in intron 9 of the KCNQ1 gene and is read as an antisense

transcript10. Kcnq1ot1 associates with the chromosomal region it was transcribed from12, leading

to in cis silencing of the maternally expressed imprinted genes Kcnq1, Cdkn1c, Slc22a18, and

Phlda211,12.

1.3.1.4.3 Intergenic Germline Differentially Methylated Region (IG-DMR)

IG-DMR is a primary parent-of-origin-specific DMR associated with parent-of-origin-specific

gene expression. IG-DMR is located between the paternally expressed imprinted gene DLK1 and

maternally expressed imprinted gene MEG3 (Meg3 in mice is called Gtl2). IG-DMR is usually

methylated in sperm and unmethylated in ova175. The developing embryo and placenta inherit

these parental methylation patterns.

In mice, a targeted deletion of murine IG-DMR on paternal chromosome 12 did not interfere

with the imprinted gene expression pattern of Dlk1, Gtl2, miR-127, miR-136, Rtl1, and Dio3;

paternally expressed imprinted genes Dlk1, Rtl1, and Dio3 were expressed and maternally

expressed imprinted genes Gtl2, miR-127, and miR-136 were silent on the paternal chromosome

12 with the targeted deletion of IG-DMR231. Mice with a targeted deletion of murine IG-DMR on

15

paternal chromosome 12 and a wild-type maternal chromosome 12 developed normally231. A

targeted deletion of murine IG-DMR on maternal chromosome 12 caused the normally silent

paternally expressed imprinted genes Dlk1, Rtl1, and Dio3 to be expressed and the normally

expressed maternally expressed imprinted genes Gtl2, miR-127, and miR-136 to be silenced on

the maternal chromosome 12 with the targeted deletion of IG-DMR231. Mice with a targeted

deletion of murine IG-DMR on maternal chromosome 12 and a wild-type paternal chromosome

12 did not survive more than a few hours after birth231.

1.3.1.4.4 DIRAS3 DMR

DIRAS3 DMR is another parent-of-origin-specific DMR associated with parent-of-origin-

specific gene expression. DIRAS3 DMR overlaps the promoter region and gene body of the

paternally expressed imprinted gene DIRAS37,232. Changes in covalent post-translational histone

tail modifications (discussed later in “Covalent Post-translational Modification of Histone Tails”

section) and DNA methylation in the promoter region of DIRAS3 can decrease or increase the

expression of DIRAS37.

DIRAS3 is paternally expressed and maternally silenced in breast tissue232. Silencing of DIRAS3

has been noted in 25-55% of breast tumours233,234. The general female population’s lifetime risk

of breast cancer is 1 in 8235,236. From the above values, it can be roughly estimated that the

general female population’s lifetime risk of breast cancer with silencing of DIRAS3 is between 1

in 32 and 11 in 160. The silencing of DIRAS3 in breast tumours has been observed in association

with a deletion of the paternal DIRAS3232.

In mice, expression of functional human DIRAS3 (achieved by generating chimeric conceptuses

by inserting embryonic stem cells expressing functional human DIRAS3 into conceptuses at the

blastocyst stage) interferes with normal growth, development of the thymus, development of the

cerebellar cortex, ovarian folliculogenesis, and mammary gland development and lactation237.

16

Figure 1-2: Imprinted Regions/Domains on Human Chromosome 11p15

A simple representation of imprinted regions/domains on human chromosome 11p15 on maternally (MAT) and

paternally (PAT) inherited human chromosomes. Maternal and paternal chromosomes are usually inherited from ova

and sperm respectively. Maternally expressed imprinted genes are shown as red arrows and paternally expressed

imprinted genes are shown as blue arrows. Light red and light blue bars show corresponding silent imprinted genes.

IC1 (H19DMR) and IC2 (KvDMR1) are the imprinting centres for the imprinted regions/domains on human

chromosome 11p15. Black-filled squares represent DNA methylated regions and white-filled squares represent

unmethylated DNA regions. From the figure, it can be observed that IC1 is paternally methylated and IC2 is

maternally methylated. Drawing is not to scale. Adapted from Developmental Biology, Vol. 320, Guo et al., Altered

gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA)

placentae, 79-91, Copyright (2008), with permission from Elsevier.

17

Figure 1-2: Imprinted Regions/Domains on Human Chromosome 11p15

18

1.3.2 Covalent Post-translational Modification of Histone Tails

Chromatin is composed of DNA wrapped around histone nucleosome cores (Figure 1-3)238,239.

Approximately 147 base pairs of DNA wrap around a single nucleosome core240. The

nucleosome octamer core is composed of two H2A-H2B dimers and one H3-H4 tetramer239.

Histone H2A, H2B, H3, and H4 have long amino acid tails, which extend out from the globular

core, that can be covalently post-translationally modified241. These covalent post-translational

histone tail modifications, that include phosphorylation, acetylation, and methylation (Figure 1-

4)242,243, may work separately or together to cause downstream effects, such as signal activation

and/or enhancing/repressing gene expression of the local genomic region241.

Some proteins can bind to specific covalent post-translational histone tail modifications. These

proteins may affect RNA polymerase recruitment, which in turn may lead to changes in gene

expression. For example, transcriptional repressor Polycomb (PcG) complex PRC1 can bind to

trimethylated histone 3 lysine 27 (H3K27)244,245. It has been suggested PRC1 may inhibit

transcription by blocking the binding of key transcription factors in promoter/enhancer genomic

regions, RNA polymerase binding to gene promoters, transcription initiation, and/or transcription

elongation246.

Examples of covalent post-translational histone tail modifications that repress transcription are

histone 3 lysine 9 (H3K9) deacetylation, histone 3 lysine 4 (H3K4) demethylation, histone 3

lysine 27 (H3K27) methylation, H3K9 methylation, and histone 4 lysine 20 (H4K20)

methylation247-250. Reversing the covalent post-translational histone tail modifications mentioned

above enhances transcription.

19

Figure 1-3: Chromatin: DNA and its Associated Proteins

A simple representation of DNA, which contains methylated and unmethylated bases, wrapped around histones.

Histones have long amino acid tails, which extend out from the globular core. “Me” indicates a methylated base.

Adapted by permission from Nature Publishing Group: Nature (Qiu, J. (2006). "Epigenetics: unfinished symphony."

Nature 441(7090): 143-145), copyright (2006).

20

Figure 1-3: Chromatin: DNA and its Associated Proteins

21

Figure 1-4: Location of Covalent Post-translational Histone Tail Modifications

Some amino acids of histone tails are subject to covalent modifications. Some of these covalent modifications

include acetylation, methylation, phosphorylation, and ubiquitylation. Adapted by permission from Nature

Publishing Group: Nature (Spivakov, M. and A. G. Fisher (2007). "Epigenetic signatures of stem-cell identity." Nat

Rev Genet 8(4): 263-271), copyright (2007).

22

Figure 1-4: Location of Covalent Post-translational Histone Tail Modifications

23

1.3.3 Long Non-coding RNAs (ncRNAs)

Long non-coding RNAs (ncRNAs) are usually functional transcripts greater than 200 nucleotides

in length251. Approximately 35,000 long non-coding RNAs (ncRNAs) have been identified in the

mouse genome252. Long non-coding RNAs (ncRNAs) can have roles in regulating the expression

pattern of imprinted genes, regulating the activation of transcription factors, interfering with

transcription factor binding, regulating splicing during transcription, interfering with translation,

and X-inactivation253-259. Examples of long non-coding RNAs include XIST and KCNQ1OT1.

XIST is expressed from X chromosomes that are going to become inactive260. XIST coats the

chromosome it is expressed from; thereby leading to that chromosome’s silencing260. While an X

chromosome is being coated by XIST, its histone tails undergo specific covalent post-

translational modifications to strengthen the association of histones to the DNA to repress gene

expression260. KCNQ1OT1 is paternally expressed from human chromosome 11p15 and in cis

associates with it (i.e., paternal human chromosome 11p15), thereby leading to the silencing of

maternally expressed imprinted genes KCNQ1, KCNQ1DN, CDKN1C, SLC22A18, and PHLDA2

on paternal human chromosome 11p1511,12,261,262.

1.3.4 MicroRNAs (miRNAs)

MicroRNAs (miRNAs) are involved in some aspects of epigenetic regulation. MicroRNAs

(miRNAs) are 20 to 25 nucleotides in length and interact with protein complexes to degrade and

inhibit translation of complementary RNA targets263,264. MicroRNAs (miRNAs) are predicted to

regulate 60% of the genes in the human genome265. Some microRNAs are involved in cell

growth, proliferation, and differentiation266-268. For example, miR-124a may hinder the

differentiation of human embryonic stem cells (hESCs)269.

1.3.5 Crosstalk Between Epigenetic Mechanisms

Different epigenetic mechanisms can interact with each other to refine epigenetic control of gene

expression. For example, DNA methylation and repressive covalent post-translational histone tail

24

modifications silence the miR200 family of miRNAs, which impede epithelial to mesenchymal

transition (EMT) and tumour invasion270-275.

Methylated cytosines can be recognized by methyl-cytosine binding proteins MeCP2, MBD1,

and MBD2276. These methyl-CpG binding proteins can interact with complexes of proteins, such

as HDAC1 and Sin3A, to covalently modify histone tails to loosen or tighten chromatin to affect

gene expression277-280. MeCP2 may interact with a methyltransferase for H3K9 (SUV39H1),

histone tail deacetylases HDAC1 and HDAC2 to tighten chromatin and inhibit gene

expression281-287. H3K9 methylation is recognized by heterochromatin protein 1 (HP1), which

can interact with DNA methyltransferase DNMT3a, which can methylate the DNA in the

region288-290. As can be seen with the examples above, different epigenetic mechanisms can

regulate each other. Some may even form feedback loops to enable the refinement of epigenetic

regulation.

25

1.4 Human Disorders Associated With Uniparental Origin Of Limited Genomic Regions

Beckwith-Wiedemann syndrome (BWS), Russell-Silver syndrome (RSS), Prader-Will syndrome

(PWS), and Angelman syndrome (AS) are examples of human genetic/genomic disorders

associated with imprinted regions that may exhibit localized genomic areas (i.e., all or part of

one chromosome) of uniparental origin.

1.4.1 Beckwith-Wiedemann syndrome (BWS)

Beckwith-Wiedemann syndrome (BWS) is a complex genetic/genomic overgrowth disorder.

Features of BWS include macroglossia, exomphalos, gigantism, hemihyperplasia, facial nevus

flammeus, prominent eyes with infra-orbital creases, midfacial hypoplasia, anterior earlobe

creases and posterior helical pits206,291-294. Visceromegaly, abdominal wall defects, and cardiac

malformations may also occur206,291,293,295,296. Individuals with BWS are at a higher risk for

embryonal cancers206,297,298. The incidence of BWS has been estimated to be one in 13,700

individuals206. In ~20% of cases, BWS is caused by a paternal uniparental disomy for human

chromosome 11p15 (pUPD11p15)206,299. Other molecular causes of BWS involving human

chromosome 11p15 include: a loss of DNA methylation at IC2 (~50% of cases), a mutation in

the maternal copy of the maternally expressed imprinted gene CDKN1C (~10% of cases), a gain

of DNA methylation at IC1 (~2%-7% of cases), a duplication of the chromosome 11p15 region

(~1% of cases), and/or a translocation/inversion involving chromosome 11p15 (~1% of

cases)206,299-302. The mechanism of BWS for approximately 14% of cases is unknown206,299.

1.4.2 Russell-Silver syndrome (RSS)

Russell-Silver syndrome (RSS) is a complex genetic/genomic undergrowth disorder. Features of

RSS include prenatal and postnatal growth retardation, hemihyperplasia, and fifth finger

clinodactyly303,304. The incidence of RSS has been estimated to be one in 3,000 - 100,000

individuals305,306. In ~30% of cases, RSS is caused by a loss of DNA methylation at IC1 on

human chromosome 11p15207,307-316. In ~10% of cases, RSS is caused by a maternal uniparental

26

disomy for human chromosome 7 (mUPD7) (~10% of cases)207,307-316. The mechanism of RSS

for approximately 60% of cases is unknown207.

1.4.3 Prader-Willi syndrome (PWS)

Prader-Willi syndrome (PWS) is a complex genetic/genomic disorder with mental and physical

abnormalities. The physical abnormalities of PWS include infantile hypotonia, early childhood

obesity, small hands and feet, hypogenitalism/hypogonadism, growth hormone deficiency, short

stature, sticky saliva, enamel hypoplasia, and a typical PWS face317-319. The typical PWS face has

an upturned-short nose, narrow bifrontal diameter, almond-shaped eyes, and a triangular

mouth317-319. PWS symptoms may include temper tantrums and skin picking317-319. The incidence

of PWS has been estimated to be one in 10,000 - 20,000 individuals, with a higher rate in

Caucasians317. In ~20% to ~25% of cases, PWS is caused by a maternal uniparental disomy for

human chromosome 15q11-q13 (mUPD15q11-q13)208. Other molecular causes of PWS on

human chromosome 15q11-q13 include: a deletion (~70% of cases), a defect in an imprinting

centre (2%-5% of cases), and/or a chromosomal translocation involving chromosome 15q11-q13

(<1% of cases)208,320-322.

1.4.4 Angelman syndrome (AS)

Angelman syndrome (AS) is a complex genetic/genomic disorder with neurodevelopmental

deficits. The phenotype of AS may include limb tremors, gait ataxia, severe developmental

delay, speech impairment, and frequent laughing and smiling, as well as excitability323-325. The

incidence of AS has been estimated to be one in 12,000 - 20,000 individuals326-328. In ~5% of

cases, AS is caused by a paternal uniparental disomy for human chromosome 15q11-q13

(pUPD15q11-q13)209; the reciprocal uniparental disomy, mUPD15q11-q13, causes Prader-Willi

syndrome (PWS)208. Other molecular etiologies for AS on human chromosome 15q11-q13

include: a deletion (~70% of cases), a defect in an imprinting centre (~2% of cases), a mutation

in the maternal copy of the maternally expressed imprinted gene UBE3A (~20% of cases), and/or

a chromosomal translocation involving chromosome 15q11-q13 (<1% of cases)209,329-334.

27

1.5 Previous Methods Used to Find Candidate Imprinted Genes and Candidate Parent-of-Origin-Specific Differentially Methylated Regions (DMRs) in Humans

Many approaches have been taken to identify candidate imprinted genes and candidate parent-of-

origin-specific differentially methylated regions (DMRs) in humans335-341. Candidate imprinted

genes have been identified by searching the genome for genes with DNA sequence features

around known imprinted genes. Candidate imprinted genes have also been identified by

searching the genome for genes with differential allelic expression. Candidate parent-of-origin-

specific DMRs have been identified by comparing CpG methylation among tissues of

uniparental origin, tissues with cells containing maternal/paternal uniparental disomies, and

diandric & digynic triploid placentas. These approaches are described in further detail below.

1.5.1 Searching for Candidate Parent-of-Origin-Specific DMRs Using Uniparental Tissues (AnCHMs and MCTs)

A previous study searched for candidate parent-of-origin-specific DMRs using CpG methylation

sensitive and CpG methylation insensitive restriction enzymes on genomic DNA from two

uniparental tissues (AnCHM (androgenetic complete hydatidiform mole) and MCT (mature

cystic ovarian teratoma)), followed by cloning and sequencing335. The study identified 12 DMRs,

of which only one was a known parent-of-origin-specific DMR. This DMR in particular is

maternally methylated and associated with known paternally expressed imprinted genes PLAGL1

and HYMAI34,162,184,188. Their method could not detect 18 other known parent-of-origin-specific

DMRs (13 maternally methylated, 5 paternally methylated) in the human genome that associate

with known imprinted genes18,21,23,24,26,28,163,181,182,205,335,339,342-346. Perhaps their method could not

detect the other known parent-of-origin-specific DMRs (associated with known imprinted genes)

because they were using site-specific CpG methylation sensitive and CpG methylation

insensitive restriction enzymes. The CpG methylation sensitive and CpG methylation insensitive

restriction enzymes they were using recognized the DNA sequence CCGG, which comprises a

very small proportion of the human genome. Another possible reason for their method being

unable to detect the other known parent-of-origin-specific DMRs (associated with known

imprinted genes) is aberrant CpG methylation (perhaps some known maternally methylated

28

DMRs are not completely methylated in MCTs, maybe some known paternally methylated

DMRs are not completely methylated in AnCHMs, possibly some known maternally methylated

DMRs are not completely unmethylated in AnCHMs, perchance some known paternally

methylated DMRs are not completely unmethylated in MCTs).

One of the newly identified candidate maternally methylated DMRs was in the promoter region

of TCEB3C. They utilized four fetuses with an informative transcribed polymorphism for

TCEB3C for allelic expression analyses. They observed preferential expression of TCEB3C from

the maternal chromosome in fetal lung, brain, placenta, and spinal cord. They did not observe

preferential maternal/paternal expression of TCEB3C in fetal liver or intestine. These results

suggested TCEB3C is an imprinted gene.

1.5.2 Searching for Candidate Imprinted Genes Using DNA Sequence Features Around Known Imprinted Genes

In another previous study, candidate imprinted genes were searched for using computer

algorithms to search the genome for genes with DNA sequence features around known imprinted

genes336. Their method could detect all 40 known imprinted genes that they used to optimize

their computer algorithm. Their method could not detect 27 other known imprinted genes not

included in their training set. These results may have been due to overfitting the model to the

data. Perhaps some unidentified imprinted genes do not have many, if any at all, DNA sequence

features common to known imprinted genes. They identified 156 candidate imprinted genes,

including DLGAP2 and KCNK9. They used eight fetuses with an informative transcribed

polymorphism for DLGAP2 and nine fetuses with an informative transcribed polymorphism for

KCNK9 for allelic expression analyses. They observed preferential expression of DLGAP2 from

the paternal chromosome in fetal testis and preferential expression of KCNK9 from the maternal

chromosome in fetal brain. These results suggested DLGAP2 and KCNK9 are imprinted genes.

Notably, a similar approach was used to identify candidate imprinted genes in mice347.

29

1.5.3 Searching for Candidate Imprinted Genes by Searching for Genes with Differential Allelic Expression

Another study searched for candidate imprinted genes using SNP microarrays to search for genes

with differential allelic expression in peripheral blood leukocytes (PBLs)337. They found PEG10

and ATP10A as the only known imprinted genes to have differential allelic expression in their

analyses using PBLs. They found imprinted gene PLAGL1 to not exhibit differential allelic

expression in PBLs, which was expected as Kamiya et al. (2000)188 observed expression of

imprinted gene PLAGL1 from both the maternal and paternal chromosome in peripheral blood

leukocytes (PBLs). They could not assess differential allelic expression for 64 other known

imprinted genes. This may have been due to the rarity of informative transcribed SNPs and

excluding genes with differential allelic expression that had an association with the informative

transcribed SNP. They identified 61 candidate imprinted genes, including TBC1D4 and ZNF331.

They utilized seven siblings with an informative transcribed polymorphism for TBC1D4 and six

siblings with an informative transcribed polymorphism for ZNF331 for allelic expression

analyses. They observed preferential expression of TBC1D4 from the maternal chromosome and

preferential expression of ZNF331 from the paternal chromosome in lymphoblastoid cell lines

(LCLs). They proposed TBC1D4 and ZNF331 are imprinted genes. Interestingly, other studies

have focused on differential allelic expression in humans348-353. Notably, similar approaches have

been used to identify candidate imprinted genes in mice4,354.

1.5.4 Searching for Candidate Imprinted Genes by Searching for RNA Polymerase Bound to Only One of Two Alleles

In another study, candidate imprinted genes were searched for by searching for genes with RNA

polymerase bound to only one of their two alleles (one allele on each homologous chromosome)

in a human lung fibroblast cell line (IMR90)338. They identified RNA polymerase bound to only

one of two alleles for known imprinted genes KCNQ1, PLAGL1, SNRPN, ZNF331, and ZNF597.

They could not identify RNA polymerase bound to only one of two alleles for 64 other known

imprinted genes. This may have been due to the rarity of informative transcribed SNPs. They

identified RNA polymerase bound to only one of two alleles for (excluding known imprinted

30

genes) 234 RefSeq genes, 16 microRNAs (miRNAs), and two small nucleolar RNAs

(snoRNAs). These RefSeq genes, microRNAs, and snoRNAs are candidate imprinted genes.

1.5.5 Searching for Candidate Parent-of-Origin-Specific DMRs Using Tissue with Cells Containing Maternal/Paternal Uniparental Disomies for Human Chromosome 15

A recent study searched for candidate parent-of-origin-specific DMRs on human chromosome 15

using DNA methylation profiling (using MeDIP-chip) of blood tissues with cells containing

maternal/paternal uniparental disomies for human chromosome 15339. The study identified 25

candidate parent-of-origin-specific DMRs and 4 known maternally methylated DMRs associated

with known imprinted gene SNRPN183,355,356. Notably, of the 25 candidate parent-of-origin-

specific DMRs, only 8 were confirmed using sodium bisulfite cloning/sequencing. Remarkably,

of the 8 new DMRs (all candidate maternally methylated DMRs), two DMRs are associated with

known imprinted gene MAGEL2, two DMRs are associated with known imprinted gene NDN,

two DMRs are associated with known imprinted gene SNRPN, one DMR is associated with

GABRG3, and one DMR is associated with IGF1R. Interestingly, the DMRs associated with

GABRG3 and IGF1R are almost completely methylated on the maternal chromosome and almost

completely unmethylated on the paternal chromosome. Allelic expression analyses of IGF1R in

cord blood and term placenta did not reveal any instances of preferential allelic expression.

These results suggested IGF1R is not imprinted in cord blood or term placenta.

1.5.6 Searching for Candidate Parent-of-Origin-Specific DMRs Using Biparental Tissues and Tissues with Cells Containing Genome-Wide Maternal/Paternal Uniparental Disomies

In another recent study, candidate parent-of-origin-specific DMRs were searched for using CpG

methylation profiling (using the Illumina Infinium Human Methylation27 promoter microarray)

of biparental tissues (blood, placenta, brain, muscle, fat, and buccal cells) and blood tissues with

cells containing genome-wide maternal/paternal uniparental disomies340. The study identified 2

candidate paternally methylated CpG sites (in blood, placenta, brain, muscle, fat, and buccal

cells) and 12 candidate maternally methylated CpG sites (in blood, placenta, brain, muscle, fat,

31

and buccal cells). Notably, of the 14 candidate parent-of-origin-specific differentially methylated

CpG sites (DMCpGs), only 2 candidate paternally methylated CpG sites and 7 candidate

maternally methylated CpG sites were confirmed using sodium bisulfite cloning/sequencing.

Interestingly, the 2 candidate paternally methylated CpG sites are associated with known

maternally expressed imprinted gene ZNF597, 5 candidate maternally methylated CpG sites are

associated with known imprinted gene RB1, and 2 candidate maternally methylated CpG sites are

associated with FAM50B. Allelic expression analysis of FAM50B in one placenta sample

revealed preferential allelic expression. This result suggested FAM50B may be an imprinted

gene. Interestingly, Zhang et al. (2011)357 observed preferential paternal expression of FAM50B

in four fetal conceptuses.

1.5.7 Searching for Candidate Parent-of-Origin-Specific DMRs Using Biparental Diploid Placentas, Diandric Triploid Placentas, Digynic Triploid Placentas, and Androgenetic Complete Hydatidiform Moles (AnCHMs)

A relatively new study searched for candidate parent-of-origin-specific DMRs using CpG

methylation profiling (using the Illumina Infinium Human Methylation27 promoter microarray)

of biparental diploid (one maternal genome and one paternal genome) placentas, diandric triploid

(two paternal genomes and one maternal genome) placentas, digynic triploid (2 maternal

genomes and 1 paternal genome) placentas, and androgenetic complete hydatidiform moles

(AnCHMs)341. The study identified 40 paternally methylated CpG sites (known and candidate)

and 68 maternally methylated CpG sites (known and candidate) that are associated with 26 and

37 genes respectively. Notably, 4 candidate maternally methylated CpG sites were located in the

promoter region of known paternally expressed imprinted gene L3MBTL1, one candidate

maternally methylated CpG site was located in the promoter region of known paternally

expressed imprinted gene NAP1L5, one candidate paternally methylated CpG site was located in

the promoter region of known maternally expressed imprinted gene ZNF597, one candidate

paternally methylated CpG site was located in the promoter region of known maternally

expressed imprinted gene CDKN1C, and one candidate maternally methylated CpG site was

located in the promoter region of candidate imprinted gene FAM50B. Monoallelic CpG

methylation patterns were observed in the candidate maternally methylated DMR associated with

32

candidate imprinted gene FAM50B. Allelic expression analyses of FAM50B in five different

placenta samples revealed preferential paternal expression of FAM50B.

33

1.6 Hypothesis and Aims

I hypothesize that candidate parent-of-origin-specific differentially methylated CpG sites

(DMCpGs), some of which may reside in parent-of-origin-specific DMRs associated with nearby

known and/or novel imprinted genes in humans, can be identified by comparing the CpG

methylation of individual CpG sites in human uniparental and biparental tissues. These unique

human uniparental tissues are androgenetic complete hydatidiform mole (AnCHM) and mature

cystic ovarian teratoma (MCT). To test my hypothesis, the following aims were developed:

1) To analyze CpG methylation using sodium bisulfite-treated DNA from human

uniparental and biparental tissues on Illumina Infinium Human Methylation27 promoter

microarrays. The CpG methylation in known paternally and maternally methylated

DMRs (associated with known imprinted genes) will be characterized using statistical

values. These statistical values will be used to set thresholds/criteria to identify candidate

parent-of-origin-specific DMCpGs. Some of these candidate parent-of-origin-specific

DMCpGs may reside in parent-of-origin-specific DMRs associated with nearby known

and/or novel imprinted genes

2) To use targeted quantitative sodium bisulfite pyrosequencing to assess the CpG

methylation around selected candidate parent-of-origin-specific DMCpGs identified by

comparing CpG methylation using sodium bisulfite-treated DNA from human uniparental

and biparental tissues on Illumina Infinium Human Methylation27 promoter microarrays

3) To determine the allelic expression pattern of a candidate novel imprinted gene in

humans, AXL, which has a candidate parent-of-origin-specific DMCpG in its promoter

region identified by comparing CpG methylation using sodium bisulfite-treated DNA

from human uniparental and biparental tissues on Illumina Infinium Human

Methylation27 promoter microarrays

35

Chapter 2 Materials and Methods

2 Materials and Methods

2.1 Sample Collection

Blood samples were collected from individuals undergoing routine blood tests after informed

consent. DNA was extracted from these samples using a standard phenol/chloroform extraction

process210,358,359. Blood samples were collected mostly from children (Appendices Table A-2).

The placenta samples were provided by the Biobank at Mount Sinai Hospital. DNA was

extracted from these samples (as described previously)210,358. Placenta samples were collected

from conceptuses older than 24 weeks gestation (Appendices Table A-3).

One androgenetic complete hydatidiform mole (AnCHM) sample was obtained from Mount

Sinai Hospital. Two other AnCHM samples were obtained from Montreal Children’s Hospital.

One mature cystic ovarian teratoma (MCT) sample was obtained from the Biopathology Center,

CHTN Pediatric Division, Nationwide Children’s Hospital, Columbus, Ohio. DNA was extracted

from these samples (as described previously)166,210,358.

DNA was obtained from the following: 1 lymphoblastoid cell line (LCL) derived from a patient

with paternal UPD11p15 (pUPD11p15), 1 blood sample from a patient with maternal UPD14

(mUPD14), 1 blood sample from a patient with paternal UPD14 (pUPD14), and 1

lymphoblastoid cell line (LCL) derived from a patient with paternal UPD4 (pUPD4).

DNA samples were stored at 4°C.

2.2 Sodium Bisulfite Treatment of DNA

All DNA samples were sodium bisulfite-treated using the EpiTect Bisulfite kit (Qiagen). Sodium

bisulfite treatment enables the quantification of cytosine methylation for individual cytosines in

DNA. The individual steps in the deamination of cytosine using sodium bisulfite treatment are

shown in Figure 2-1360.

36

Figure 2-1: Steps in the Sodium Bisulfite-Mediated Deamination of Cytosine

Cytosine undergoes a sulphonation reaction to produce cytosine sulphonate361,362. Cytosine sulphonate undergoes a

hydrolytic deamination reaction to produce uracil sulphonate361,362. Uracil sulphonate undergoes an alkali

desulphonation reaction to produce uracil361,362. Adapted by permission from Nature Publishing Group: Nature

Protocols (Clark, S. J., A. Statham, et al. (2006). "DNA methylation: bisulphite modification and analysis." Nat

Protoc 1(5): 2353-2364), copyright (2006).

37

Figure 2-1: Steps in the Sodium Bisulfite-Mediated Deamination of Cytosine

38

2.3 Microarray Processing

The sodium bisulfite-treated DNA for 16 blood samples, 5 placenta samples, 3 AnCHM samples,

1 MCT sample, and 1 paternal UPD4 lymphoblastoid cell line (LCL) sample were sent to The

Centre for Applied Genomics (TCAG) to be prepared for Illumina Infinium Human

Methylation27 promoter microarrays. For each sample, the Illumina microarray assesses the

proportion of CpG methylation at 27,578 individual CpG sites. More than 14,000 RefSeq gene

promoters are covered by these 27,578 individual CpG sites, with an average of 2 CpG sites per

gene promoter. Cancer-related and imprinted genes have more extensive coverage (three to

twenty CpG sites per gene).

The Illumina microarray uses two site-specific probes (both 50 base pairs long) for each targeted

individual CpG site. One site-specific probe targets the unmethylated CpG site. The other site-

specific probe targets the methylated CpG site. The targeted CpG site is situated to the end of

the 50 base pair oligonucleotide (not the end with the bead) to enable quantification of cytosine

methylation by incorporating a fluorescently labeled ddNTP onto the 50 base pair

oligonucleotide.

The single measure (one-way random) intraclass correlation coefficient (ICC) for a technical

replicate (same sodium bisulfite treatment for a sample) run on the microarray was .98

(calculated using computer application SPSS Statistics 17.0 and all 27,578 Illumina microarray

CpG sites). It was important for the technical replicate to be from the same sodium bisulfite

treatment because using a different sodium bisulfite treatment may have introduced variability.

The single measure (one-way random) intraclass correlation coefficient (ICC) was chosen

because the same sample was run twice on the same type of microarray.

Within the TCAG facility, the sodium bisulfite-treated DNAs underwent whole-genome

amplification (WGA), enzymatic fragmentation, and hybridization to the Illumina Infinium

Human Methylation27 promoter microarray. Probes underwent single-base extension using

fluorescently labeled ddNTPs. A probe was only extended with a fluorescently labeled ddNTP

when its end (not the end attached to the bead) was bound to the sodium bisulfite-treated DNA.

The microarray was washed, stained, and scanned. The scanned image underwent feature

extraction. I received the feature extracted data from TCAG as “.idat” files.

39

2.4 CpG Methylated Proportion and Detection P-value For Each Targeted CpG Site

The feature extracted data was loaded into Illumina’s Beadstudio 3 computer application. In

Beadstudio 3, samples were organized into groups (3 AnCHMs in one group, 16 bloods in one

group, 1 MCT in one group, 5 placentas in one group, 1 pUPD4 in one group). The average

normalization (across chips) setting was chosen in BeadStudio 3 because it allowed for the

comparison of samples run on different microarray chips. Using these steps, BeadStudio 3

calculated the CpG methylated proportion and detection p-value for each individual microarray

CpG site in each sample. The detection p-value showed how significantly different the signal for

the CpG site was when compared to the background signal. This data was uploaded to NCBI’s

(The National Center for Biotechnology Information’s) GEO (Gene Expression Omnibus) and

was given the accession number GSE22091.

CpG methylated proportions were replaced with a “N/A” if their CpG site detection p-values

were greater than .05 (i.e., the signal intensity for the CpG site was not significantly different

from the background). This was done because if the signal intensity for the CpG site is not

significantly different from the background, the CpG site’s calculated CpG methylated

proportion is unreliable. The number of CpG methylated proportions replaced with “N/A” varied

between samples (Appendices Table A-4).

To convert a CpG methylated proportion value into a percent CpG methylation value; multiply

the CpG methylated proportion value by 100 to get the percent CpG methylation value.

2.5 Statistical Analyses to Characterize CpG Methylation

In Illumina Beadstudio 3, a Mann-Whitney statistical test comparing CpG methylation between

AnCHM samples and blood samples was performed for each individual microarray CpG site. I

chose the AnCHMs instead of the MCT to be the uniparental group for comparison because I had

more AnCHMs than MCTs (3 AnCHMs, 1 MCT). I chose the bloods instead of the placentas to

be the biparental group because I had more bloods than placentas (16 bloods, 5 placentas). Since,

to my knowledge at the time, no one had noted that the CpG methylation data in known parent-

of-origin-specific DMRs (associated with known imprinted genes) is normally distributed, I

40

chose to utilize the Mann-Whitney statistical test because it can “detect differences in shape and

spread as well as just differences in medians”363 and does not require the underlying data to be

normally distributed.

The maximum p-value (not adjusted using the false discovery rate (FDR)) on the list less than

.05 has an associated q-value (“the minimum FDR that can be attained when calling that feature

significant”364) less than .06. These results may suggest that adjusting p-values using the FDR

may not have been necessary when using a p-value cutoff of .05. As a side note, the maximum p-

value (not adjusted using the false discovery rate (FDR)) on the list less than .01 has an

associated q-value of approximately .013. I decided that it would be better to adjust the p-values

using the FDR to be on the safe side. Also as a side note, the maximum p-value (adjusted using

the FDR) on the list less than .05 has an associated q-value of approximately .022 (this q-value

was determined based on the unadjusted p-value distribution). Also, the maximum p-value

(adjusted using the FDR) on the list less than .01 has an associated q-value of approximately .004

(this q-value was also determined based on the unadjusted p-value distribution). Notably, there

are more than expected unadjusted and FDR-adjusted p-values less than .05 (Figure 2-2).

All the data (e.g., p-value for the Mann-Whitney test mentioned above (comparing AnCHMs to

bloods), CpG methylated proportion per targeted CpG site per sample, detection p-value, each

targeted CpG site’s TargetID, the location of the CpG site in the human genome (NCBI Build

36), the gene promoter the CpG site is in, if the CpG site is in a CpG island) from BeadStudio 3

was exported to Microsoft Excel 2007 in “.csv” format. The data pertaining to the sex

chromosomes (1092 of 27578 Illumina microarray CpG sites) were deleted from the “.csv” file.

This was done because I decided to focus on autosomal loci. To focus on X chromosome loci, an

alternative method would have been required. This would have been necessary because males

usually have one X chromosome and females usually have two X chromosomes. Also, the X

chromosome in males would usually be inherited from the mother, whereas in females, one X

chromosome would usually be inherited from each parent. Also, X chromosome cytosine

methylation could be altered by X inactivation in females.

In Microsoft Excel 2007, I calculated values that would be used to characterize CpG methylation

in known parent-of-origin-specific DMRs (associated with known imprinted genes). These

values would later be used to set thresholds/criteria to find candidate parent-of-origin-specific

41

differentially methylated CpG sites (DMCpGs), some of which may reside in parent-of-origin-

specific DMRs associated with nearby known and/or novel imprinted genes. The median CpG

methylated proportions for each CpG site for the AnCHMs, bloods, and placentas was

calculated. The median instead of the mean was calculated because a normal distribution could

not be assumed. The CpG methylated interquartile ranges (IQRs) for each CpG site for the

AnCHMs, bloods, and placentas was calculated. IQRs were calculated because IQRs measure

variability and they are usually associated with medians. The CpG methylated proportion

difference between the (blood median and AnCHM median) and the (placenta median and

AnCHM median) for each CpG site was computed. The CpG methylated proportion differences

for each CpG site when comparing one of the AnCHM to the MCT was also computed. It is

expected that AnCHMs and MCTs have opposite CpG methylation values in parent-of-origin-

specific DMRs associated with imprinted genes. It was decided that the AnCHM with the highest

CpG methylation difference when compared to the MCT in known parent-of-origin-specific

DMRs (associated with known imprinted genes) would be the one considered to be the best

AnCHM to use for comparisons. This difference was inferred using the intraclass correlation

coefficient (ICC). Intraclass correlation coefficients (ICCs) measure the amount of agreement

between sets of paired data. The ICCs were calculated using the computer application SPSS

Statistics 17.0. The absolute CpG methylated proportion difference between the (blood median -

AnCHM median) column and the (MCT – AnCHM (best)) column for each CpG site was

computed. The difference between the MCT and the best AnCHM was expected to be greater

than the difference between the blood median and AnCHM median. The absolute CpG

methylated proportion difference between the (placenta median - AnCHM median) column and

the (MCT – AnCHM (best)) column for each CpG site was also computed. The difference

between the MCT and the best AnCHM was expected to be greater than the difference between

the placenta median and AnCHM median.

For CpG sites located in parent-of-origin-specific DMRs (associated with imprinted genes), it is

expected the blood and placenta (both have one sperm-derived genome and one oocyte-derived

genome) will have median CpG methylation values in between the AnCHMs (two sperm-derived

genomes) and MCT (two oocyte-derived genomes). In paternally methylated DMRs associated

with imprinted genes, it is expected AnCHMs have 100% CpG methylation, MCTs have 0%

CpG methylation, bloods and placentas have 50% CpG methylation (Figure 2-3). In maternally

42

methylated DMRs associated with imprinted genes, it is expected AnCHMs have 0% CpG

methylation, MCTs have 100% CpG methylation, bloods and placentas have 50% CpG

methylation (Figure 2-3). In my experiment, in the known paternally methylated DMR

(associated with known imprinted gene GNAS), GNAS NESP55 DMR, I can observe AnCHMs

with ~87% CpG methylation, the MCT with ~11% CpG methylation, bloods with ~42% CpG

methylation, and placentas with ~55% CpG methylation.

2.6 Compiled List of Microarray CpG Sites in Known Parent-of-Origin-Specific DMRs Associated With Known Imprinted Genes

A list of Illumina Infinium Human Methylation27 promoter microarray CpG sites located in

known parent-of-origin-specific DMRs (associated with known imprinted genes) was compiled

(these CpG sites are listed in Appendices Table A-5) using the following journal articles: Yuan

et al. (2003)18, Riemenschneider et al. (2008)19, Valleley et al. (2010)342, Arima et al. (2006)179,

Kamiya et al. (2000)188, Bliek et al. (2009)365, Arnaud et al. (2003)21, Kainz et al. (2007)22, Monk

et al. (2008)23, Riesewijk et al. (1997)24, Li et al. (2002)343, Frevel et al. (1999)172, Beatty et al.

(2006)205, Zeschnigk et al. (1997)26, Dasoula et al. (2007)366, Murphy et al. (2001)27, Liu et al.

(2000)28, and Bastepe et al. (2001)189. Only one (out of the 46) Illumina Infinium Human

Methylation27 promoter microarray CpG sites located in known parent-of-origin-specific DMRs

(associated with known imprinted genes) was excluded from the list. This particular CpG site

(cg06191076) did not have blood and placenta median CpG methylation values in between the

AnCHMs and MCT (the blood and placenta were more heavily methylated than the AnCHMs

and MCT).

43

Figure 2-2: Distribution of Unadjusted and FDR-adjusted Mann-Whitney P-values

Histograms showing the distributions of unadjusted (A) and FDR-adjusted (B) Mann-Whitney p-values. The

unadjusted p-values came from Mann-Whitney statistical tests performed for each individual microarray CpG site

comparing CpG methylation between AnCHM samples and blood samples. The unadjusted p-values were converted

into FDR-adjusted p-values using the false discovery rate (FDR).

44

A

B

Figure 2-2: Distribution of Unadjusted and FDR-adjusted Mann-Whitney P-values

45

Figure 2-3: Expected CpG Methylation in Maternally/Paternally Methylated DMRs

Androgenetic complete hydatidiform moles (AnCHMs) and mature cystic ovarian teratomas (MCTs) are uniparental

tissues. Unlike somatic tissues with a genomic contribution from each parent, AnCHMs have two paternal (i.e.,

sperm-derived) copies of each chromosome and MCTs have two maternal (i.e., oocyte-derived) copies of each

chromosome. Placenta and AnCHM arise from trophoblastic tissue. Fibroblasts compose a large proportion of

MCTs. WBC stands for white blood cell.

46

Figure 2-3: Expected CpG Methylation in Maternally/Paternally Methylated DMRs

47

2.7 Selection Criteria for Candidate Maternally Methylated CpG Sites in Blood

Minimum and maximum values were calculated for the median CpG methylated proportions for

each CpG site in KMMDMRs (known maternally methylated DMRs associated with known

imprinted genes) for the bloods; the minimum and maximum values were ~0.345 and ~0.805

respectively. A maximum value was calculated for the median CpG methylated proportions for


imprinted genes) for the AnCHMs; the maximum value was ~0.446. Maximum values were

calculated for the CpG methylated proportion interquartile ranges (IQRs) for each CpG site in

KMMDMRs (known maternally methylated DMRs associated with known imprinted genes) for

the AnCHMs and bloods; the maximum values for the AnCHMs and bloods were ~0.203 and

~0.102 respectively. A minimum value was calculated for the CpG methylated proportion

difference between the (blood median and AnCHM median) for each CpG site in KMMDMRs

(known maternally methylated DMRs associated with known imprinted genes); the minimum

value was ~0.216. A minimum value was calculated for the CpG methylated proportion

difference between the (best AnCHM and MCT) for each CpG site in KMMDMRs (known

maternally methylated DMRs associated with known imprinted genes); the minimum value was

~0.299. A minimum value was calculated for the CpG methylated proportions for CpG sites

within KMMDMRs (known maternally methylated DMRs associated with known imprinted

genes) for the MCT; the minimum value was ~0.446. All of these maximum and minimum

values were used to form the criteria for candidate maternally methylated CpG sites in blood.

The criteria also required that candidate maternally methylated CpG sites in blood have

significant (i.e., p-value < .05) Mann-Whitney statistical tests (with p-values adjusted using the

FDR) comparing the AnCHMs to bloods. In addition, the criteria also required that the difference

between the MCT and the best AnCHM be greater than the difference between the blood median

and AnCHM median for a given candidate maternally methylated CpG site in blood. With these

criteria, I identified all 43 maternally methylated CpG sites on the list of Illumina Infinium

Human Methylation27 promoter microarray CpG sites located in known parent-of-origin-

specific DMRs (associated with known imprinted genes). I also identified 365 candidate

maternally methylated CpG sites in blood (Appendices Table A-6).

48

2.8 Selection Criteria for Candidate Maternally Methylated CpG Sites in Placenta



imprinted genes) for the placentas; the minimum and maximum values were ~0.241 and ~0.801

respectively. A maximum value was calculated for the median CpG methylated proportions for


imprinted genes) for the AnCHMs; the maximum value was ~0.446. Maximum values were


KMMDMRs (known maternally methylated DMRs associated with known imprinted genes) for

the AnCHMs and placentas; the maximum values for the AnCHMs and placentas were ~0.203

and ~0.216 respectively. A minimum value was calculated for the CpG methylated proportion

difference between the (placenta median and AnCHM median) for each CpG site in KMMDMRs

(known maternally methylated DMRs associated with known imprinted genes); the minimum

value was ~0.151. A minimum value was calculated for the CpG methylated proportion

difference between the (best AnCHM and MCT) for each CpG site in KMMDMRs (known

maternally methylated DMRs associated with known imprinted genes); the minimum value was

~0.299. A minimum value was calculated for the CpG methylated proportions for CpG sites

within KMMDMRs (known maternally methylated DMRs associated with known imprinted

genes) for the MCT; the minimum value was ~0.446. All of these maximum and minimum

values were used to form the criteria for candidate maternally methylated CpG sites in placenta.

In addition, the criteria also required that the difference between the MCT and the best AnCHM

be greater than the difference between the placenta median and AnCHM median for a given

candidate maternally methylated CpG site in placenta. With these criteria, I identified all 43

maternally methylated CpG sites on the list of Illumina Infinium Human Methylation27

promoter microarray CpG sites located in known parent-of-origin-specific DMRs (associated

with known imprinted genes). I also identified 491 candidate maternally methylated CpG sites in

placenta (Appendices Table A-7).

49

2.9 Selection Criteria for Candidate Paternally Methylated CpG Sites in Blood


each CpG site in KPMDMRs (known paternally methylated DMRs associated with known

imprinted genes) for the bloods; the minimum and maximum values were ~0.418 and ~0.461

respectively. A minimum value was calculated for the median CpG methylated proportions for


imprinted genes) for the AnCHMs; the minimum value was ~0.874. Maximum values were


KPMDMRs (known paternally methylated DMRs associated with known imprinted genes) for

the AnCHMs and bloods; the maximum values for the AnCHMs and bloods were ~0.033 and

~0.057 respectively. A maximum value was calculated for the CpG methylated proportion

difference between the (blood median and AnCHM median) for each CpG site in KPMDMRs

(known paternally methylated DMRs associated with known imprinted genes); the maximum

value was ~-.427. A maximum value was calculated for the CpG methylated proportion

difference between the (best AnCHM and MCT) for each CpG site in KPMDMRs (known

paternally methylated DMRs associated with known imprinted genes); the maximum value was

~-.787. A maximum value was calculated for the CpG methylated proportions for CpG sites

within KPMDMRs (known paternally methylated DMRs associated with known imprinted

genes) for the MCT; the maximum value was ~0.104. All of these maximum and minimum

values were used to form the criteria for candidate paternally methylated CpG sites in blood. The

criteria also required that candidate paternally methylated CpG sites in blood have significant

(i.e., p-value < .05) Mann-Whitney statistical tests (with p-values adjusted using the false

discovery rate (FDR)) comparing the AnCHMs to bloods. In addition, the criteria also required

that the difference between the MCT and the best AnCHM be greater than the difference

between the blood median and AnCHM median for a given candidate differentially methylated

CpG site. With these criteria, I identified only the 2 paternally methylated CpG sites on the list of

Illumina Infinium Human Methylation27 promoter microarray CpG sites located in known

parent-of-origin-specific DMRs (associated with known imprinted genes). In other words, with

these criteria I did not identify any candidate paternally methylated CpG sites in blood. The

criteria for candidate paternally methylated CpG sites in blood were too strict (allowed less

variation) in comparison to the criteria for candidate maternally methylated CpG sites in blood

50

and the criteria for candidate maternally methylated CpG sites in placenta. This may have been

due to the scarcity of microarray CpG sites in KPMDMRs (known paternally methylated DMRs

associated with known imprinted genes). With more microarray CpG sites in KPMDMRs

(known paternally methylated DMRs associated with known imprinted genes), more variation

may have been introduced.

2.9.1 Adapted Selection Criteria for Candidate Paternally Methylated CpG Sites in Blood

Extra variation was introduced into the criteria for candidate paternally methylated CpG sites in

blood by adapting the criteria for candidate maternally methylated CpG sites in blood. The

adaptations are detailed in the paragraphs below.

If the maximum AnCHM median percent CpG methylation value calculated for CpG sites in

KMMDMRs (known maternally methylated DMRs associated with known imprinted genes) was

further from its expected value (0%) when compared to how far the minimum AnCHM median

percent CpG methylation value calculated for CpG sites in KPMDMRs (known paternally

methylated DMRs associated with known imprinted genes) was from its expected value (100%),

the candidate paternally methylated CpG site in blood criteria would use the maximum AnCHM

median percent CpG methylation calculated for CpG sites in KMMDMRs (known maternally

methylated DMRs associated with known imprinted genes) subtracted from 100% to be the

minimum AnCHM median percent CpG methylation value. This was done because I would

expect opposite AnCHM percent CpG methylation in KMMDMRs (known maternally

methylated DMRs associated with known imprinted genes) and KPMDMRs (known paternally

methylated DMRs associated with known imprinted genes).

If the minimum MCT percent CpG methylation value in KMMDMRs (known maternally

methylated DMRs associated with known imprinted genes) was further from its expected value

(100%) when compared to how far the maximum MCT percent CpG methylation value in

KPMDMRs (known paternally methylated DMRs associated with known imprinted genes) was

from its expected value (0%), the candidate paternally methylated CpG site in blood criteria

would use the minimum MCT percent CpG methylation value in KMMDMRs (known

51

maternally methylated DMRs associated with known imprinted genes) subtracted from 100% to

be the maximum MCT percent CpG methylation value. This was done because I would expect

opposite MCT percent CpG methylation in KMMDMRs (known maternally methylated DMRs

associated with known imprinted genes) and KPMDMRs (known paternally methylated DMRs

associated with known imprinted genes).

If the maximum or minimum blood median percent CpG methylation value calculated for CpG

sites in KMMDMRs (known maternally methylated DMRs associated with known imprinted

genes) was further from its expected value (50%) when compared to how far the maximum or

minimum blood median percent CpG methylation value calculated for CpG sites in KPMDMRs

(known paternally methylated DMRs associated with known imprinted genes) was from its

expected value (50%), the candidate paternally methylated CpG site in blood criteria would use

the difference between the maximum or minimum blood median percent CpG methylation value

(whichever is furthest from the expected 50%) calculated for CpG sites in KMMDMRs (known

maternally methylated DMRs associated with known imprinted genes) to the expected (50%) as

the allowable distance around the expected 50%.

If the maximum AnCHM IQR value calculated for CpG sites in KMMDMRs (known maternally

methylated DMRs associated with known imprinted genes) was greater than the maximum

AnCHM IQR value calculated for CpG sites in KPMDMRs (known paternally methylated

DMRs associated with known imprinted genes), the maximum allowable AnCHM IQR value for

candidate paternally methylated CpG sites in blood became the maximum AnCHM IQR value

calculated for CpG sites in KMMDMRs (known maternally methylated DMRs associated with

known imprinted genes).

If the maximum blood IQR value calculated for CpG sites in KMMDMRs (known maternally

methylated DMRs associated with known imprinted genes) was greater than the maximum blood

IQR value calculated for CpG sites in KPMDMRs (known paternally methylated DMRs

associated with known imprinted genes), the maximum allowable blood IQR value for candidate

paternally methylated CpG sites in blood became the maximum blood IQR value calculated for

CpG sites in KMMDMRs (known maternally methylated DMRs associated with known

imprinted genes).

52

If the minimum difference between the calculated (blood.median-AnCHM.median) for CpG sites

in KMMDMRs (known maternally methylated DMRs associated with known imprinted genes)

was smaller than the minimum difference between the calculated (AnCHM.median-

blood.median) for CpG sites in KPMDMRs (known paternally methylated DMRs associated

with known imprinted genes), the minimum difference between the calculated (AnCHM.median-

blood.median) for candidate paternally methylated CpG sites in blood became the minimum

difference between the calculated (blood.median-AnCHM.median) for CpG sites in KMMDMRs

(known maternally methylated DMRs associated with known imprinted genes).

If the minimum difference between the calculated (MCT – best AnCHM) for CpG sites in


smaller than the minimum difference between the calculated (best AnCHM - MCT) for CpG

sites in KPMDMRs (known paternally methylated DMRs associated with known imprinted

genes), the minimum difference between the calculated (best AnCHM – MCT) for candidate

paternally methylated CpG sites in blood became the minimum difference between the calculated

(MCT – best AnCHM) for CpG sites in KMMDMRs (known maternally methylated DMRs


With the adapted criteria for candidate paternally methylated CpG sites in blood, I identified 77

candidate paternally methylated CpG sites in blood (Appendices Table A-8).

2.10 Selection Criteria for Candidate Paternally Methylated CpG Sites in Placenta



imprinted genes) for the placentas; the minimum and maximum values were ~0.557 and ~0.678

respectively. A minimum value was calculated for the median CpG methylated proportions for


imprinted genes) for the AnCHMs; the minimum value was ~0.874. Maximum values were


KPMDMRs (known paternally methylated DMRs associated with known imprinted genes) for

53

the AnCHMs and placentas; the maximum values for AnCHMs and placentas were ~0.033 and

~0.053 respectively. A maximum value was calculated for the CpG methylated proportion

difference between the (placenta median and AnCHM median) for each CpG site in KPMDMRs

(known paternally methylated DMRs associated with known imprinted genes); the maximum

value was ~-.211. A maximum value was calculated for the CpG methylated proportion

difference between the (best AnCHM and MCT) for each CpG site in KPMDMRs (known

paternally methylated DMRs associated with known imprinted genes); the maximum value was

~-.787. A maximum value was calculated for the CpG methylated proportions for CpG sites

within KPMDMRs (known paternally methylated DMRs associated with known imprinted

genes) for the MCT; the maximum value was ~0.104. All of these maximum and minimum

values were used to form the criteria for candidate paternally methylated CpG sites in placenta.

In addition, the criteria also required that the difference between the MCT and the best AnCHM

be greater than the difference between the placenta median and AnCHM median for a given

candidate differentially methylated CpG site. With these criteria, I identified only the 2

paternally methylated CpG sites on the list of Illumina Infinium Human Methylation27 promoter

microarray CpG sites located in known parent-of-origin-specific DMRs (associated with known

imprinted genes). In other words, with these criteria I did not identify any candidate paternally

methylated CpG sites in placenta. The criteria for candidate paternally methylated CpG sites in

placenta were too strict (allowed less variation) in comparison to the criteria for candidate

maternally methylated CpG sites in blood and the criteria for candidate maternally methylated

CpG sites in placenta. This may have been due to the scarcity of microarray CpG sites in

KPMDMRs (known paternally methylated DMRs associated with known imprinted genes). With

more microarray CpG sites in KPMDMRs (known paternally methylated DMRs associated with

known imprinted genes), more variation may have been introduced.

2.10.1 Adapted Selection Criteria for Candidate Paternally Methylated CpG Sites in Placenta

Extra variation was introduced into the criteria for candidate paternally methylated CpG sites in

placenta by adapting the criteria for candidate maternally methylated CpG sites in placenta. The

adaptations are detailed in the paragraphs below.

54

If the maximum AnCHM median percent CpG methylation value calculated for CpG sites in


further from its expected value (0%) when compared to how far the minimum AnCHM median

percent CpG methylation value calculated for CpG sites in KPMDMRs (known paternally

methylated DMRs associated with known imprinted genes) was from its expected value (100%),

the candidate paternally methylated CpG site in placenta criteria would use the maximum

AnCHM median percent CpG methylation calculated for CpG sites in KMMDMRs (known


be the minimum AnCHM median percent CpG methylation value. This was done because I

would expect opposite AnCHM percent CpG methylation in KMMDMRs (known maternally

methylated DMRs associated with known imprinted genes) and KPMDMRs (known paternally

methylated DMRs associated with known imprinted genes).

If the minimum MCT percent CpG methylation value in KMMDMRs (known maternally

methylated DMRs associated with known imprinted genes) was further from its expected value

(100%) when compared to how far the maximum MCT percent CpG methylation value in

KPMDMRs (known paternally methylated DMRs associated with known imprinted genes) was

from its expected value (0%), the candidate paternally methylated CpG site in placenta criteria

would use the minimum MCT percent CpG methylation value in KMMDMRs (known


be the maximum MCT percent CpG methylation value. This was done because I would expect

opposite MCT percent CpG methylation in KMMDMRs (known maternally methylated DMRs

associated with known imprinted genes) and KPMDMRs (known paternally methylated DMRs


If the maximum or minimum placenta median percent CpG methylation value calculated for

CpG sites in KMMDMRs (known maternally methylated DMRs associated with known

imprinted genes) was further from its expected value (50%) when compared to how far the

maximum or minimum placenta median percent CpG methylation value calculated for CpG sites

in KPMDMRs (known paternally methylated DMRs associated with known imprinted genes)

was from its expected value (50%), the candidate paternally methylated CpG site in placenta

criteria would use the difference between the maximum or minimum placenta median percent

CpG methylation value (whichever is furthest from the expected 50%) calculated for CpG sites

55

in KMMDMRs (known maternally methylated DMRs associated with known imprinted genes)

to the expected (50%) as the allowable distance around the expected 50%.

If the maximum AnCHM IQR value calculated for CpG sites in KMMDMRs (known maternally


AnCHM IQR value calculated for CpG sites in KPMDMRs (known paternally methylated

DMRs associated with known imprinted genes), the maximum allowable AnCHM IQR value for

candidate paternally methylated CpG sites in placenta became the maximum AnCHM IQR value



If the maximum placenta IQR value calculated for CpG sites in KMMDMRs (known maternally


placenta IQR value calculated for CpG sites in KPMDMRs (known paternally methylated DMRs

associated with known imprinted genes), the maximum allowable placenta IQR value for

candidate paternally methylated CpG sites in placenta became the maximum placenta IQR value



If the minimum difference between the calculated (placenta.median-AnCHM.median) for CpG

sites in KMMDMRs (known maternally methylated DMRs associated with known imprinted

genes) was smaller than the minimum difference between the calculated (AnCHM.median-

placenta.median) for CpG sites in KPMDMRs (known paternally methylated DMRs associated

with known imprinted genes), the minimum difference between the calculated (AnCHM.median-

placenta.median) for candidate paternally methylated CpG sites in placenta became the minimum

difference between the calculated (placenta.median-AnCHM.median) for CpG sites in

KMMDMRs (known maternally methylated DMRs associated with known imprinted genes).

If the minimum difference between the calculated (MCT – best AnCHM) for CpG sites in


smaller than the minimum difference between the calculated (best AnCHM - MCT) for CpG

sites in KPMDMRs (known paternally methylated DMRs associated with known imprinted

genes), the minimum difference between the calculated (best AnCHM – MCT) for candidate

paternally methylated CpG sites in placenta became the minimum difference between the

56

calculated (MCT – best AnCHM) for CpG sites in KMMDMRs (known maternally methylated

DMRs associated with known imprinted genes).

With the adapted criteria for candidate paternally methylated CpG sites in placenta, I identified

116 candidate paternally methylated CpG sites in placenta (Appendices Table A-9).

2.11 Targeted Quantitative Sodium Bisulfite Pyrosequencing

Targeted quantitative sodium bisulfite pyrosequencing using the PyroMark Q24 was used to

assess the percent CpG methylation of four known parent-of-origin-specific DMRs (associated

with known imprinted genes) in blood, placenta, AnCHM, and MCT. These four known parent-

of-origin-specific DMRs (associated with known imprinted genes) consisted of two paternally

methylated DMRs, IC1 and IG-DMR, and two maternally methylated DMRs, IC2 and DIRAS3. I

chose to bisulfite pyrosequencing targeting regions within known intergenic parent-of-origin-

specific DMRs (associated with known imprinted genes), IC1 (also known as H19DMR) and IG-

DMR, since the Illumina Infinium Human Methylation27 promoter microarray does not target

any CpG sites within known intergenic parent-of-origin-specific DMRs (associated with known

imprinted genes). For a comparison to the known intergenic parent-of-origin-specific DMRs

(associated with known imprinted genes), I decided to do bisulfite pyrosequencing targeting

regions within known maternally methylated DMRs IC2 and DIRAS3 DMR. I specifically chose

IC2 and DIRAS3 DMR because the lab had optimized assays available for them, i.e., IC2 and

DIRAS3 DMR.

Targeted quantitative sodium bisulfite pyrosequencing using the PyroMark Q24 was performed

in an effort to assess the extent of the candidate parent-of-origin-specific DMRs in the promoter

regions of NAP1L5, ZNF597, AXL, and RIMS2. I chose to perform bisulfite pyrosequencing

targeting the promoter regions of NAP1L5 and ZNF597 because NAP1L5 and ZNF597 were the

only known imprinted genes (without known associated parent-of-origin-specific DMRs at the

time) with candidate parent-of-origin-specific differentially methylated CpG sites (DMCpGs) in

their promoter regions. I decided to carry out bisulfite pyrosequencing targeting the promoter

region of RIMS2 since one candidate maternally methylated CpG site was detected in the

promoter region of RIMS2 and this candidate maternally methylated CpG site had differential

57

CpG methylation (~50% CpG methylation) in blood and placenta, which was rare to observe for

candidate parent-of-origin-specific differentially methylated CpG sites (DMCpGs). I chose to

perform bisulfite pyrosequencing targeting the promoter region of AXL as one candidate

paternally methylated CpG site was identified in the promoter region of AXL and this candidate

paternally methylated CpG site had one of the highest CpG methylation differences between the

best androgenetic complete hydatidiform mole (AnCHM) and mature cystic ovarian teratoma

(MCT). Also, AXL overexpression had been observed in some cancers367-371.

Targeted quantitative sodium bisulfite pyrosequencing enables the quantification of cytosine

methylation at a chosen CpG site372. The first step in targeted quantitative sodium bisulfite

pyrosequencing is to treat the genomic DNA with sodium bisulfite (under specific conditions)

and then purify the resulting DNA fragments. The second step is to amplify your sodium

bisulfite-treated region of interest using Polymerase Chain Reaction (PCR) amplification. Biotin

was not directly attached to the primers; instead an M13 sequence was attached to the 5’ end of

the primers, which annealed to a Biotin-M13 Universal Primer complex. The PCR amplification

reaction incorporates thymines instead of uracils into the daughter strands. The third step is to

sequence you PCR products using pyrosequencing reactions. The pyrosequencing reaction adds

nucleotides one at a time in a designated order and the pyrosequencer measures the proportion of

nucleotides incorporated into the PCR products. A comparison of the proportion of nucleotides

incorporated provides the percent CpG methylation. The CpG methylated proportion is

determined (at a given CpG locus) by dividing the intensity of cytosine (methylated cytosine in

the genomic DNA) by the total intensity (add the intensity of cytosine (methylated cytosine in

the genomic DNA) to the intensity of thymine (unmethylated cytosine in the genomic DNA) to

get total intensity) at the given CpG locus. The percent CpG methylation is determined by

multiplying the CpG methylated proportion by 100%.

The reagents used for the PCR amplification reactions included distilled water (Hospira

Healthcare Corporation), 10*BF (Qiagen), magnesium chloride (Qiagen), dNTPs (BioBasic),

M13-Biotin Universal Primer (Integrated DNA Technologies), oligonucleotides (Integrated

DNA Technologies), HotStar Taq (Qiagen), and sodium bisulfite-treated DNA. The amounts and

concentrations of each of these reagents for the PCR amplification reactions are shown in

Appendices Table A-10. The thermal cycler programs for the PCR amplification reactions are

shown in Appendices Table A-11. The primers used for PCR amplification reactions are listed in

58

Appendices Table A-12. The primers used for pyrosequencing reactions are listed in Appendices

Table A-13.

The primers for these reactions (PCR amplification and pyrosequencing) were designed (using

the program PSQ Assay Design 1.0.6) to only amplify sodium bisulfite-converted DNA. Also,

sodium bisulfite conversion controls were included in pyrosequencing assays. These sodium

bisulfite conversion controls were cytosines not followed by guanines in the genomic DNA.

These sodium bisulfite conversion controls are expected to appear as thymines, instead of

cytosines, in the pyrosequencing reactions.

2.12 Box-and-whisker Plots

I used the computer application SPSS Statistics 18.0 to generate box-and-whisker plots. The

horizontal line inside the box-and-whisker plot is the median of the group. The top of the box is

the 75th percentile and the bottom of the box is the 25th percentile. The bottom whisker goes to

the data point above or at (25th percentile – 1.5 * interquartile range). The upper whisker goes to

the data point below or at (75th percentile + 1.5 * interquartile range). The dots outside of the

box and whiskers represent outliers and the stars represent extreme outliers (more than (3 *

interquartile range) from the box).

2.13 Sodium Bisulfite Cloning/Sequencing

Sodium bisulfite cloning/sequencing was used to assess the CpG methylation of 14 CpG sites in

a 355 base-pair fragment from the AXL promoter region in one placenta (Placenta2), one

AnCHM (AnCHM1), and the MCT sample. I chose to perform bisulfite sequencing targeting the

identified candidate paternally methylated CpG sites (within the promoter region of AXL) in an

effort to support and determine the boundaries of the candidate paternally methylated DMR

(within the promoter region of AXL). I chose to perform bisulfite sequencing targeting the

identified candidate paternally methylated CpG sites (within the promoter region of AXL)

because CpG methylation in the promoter region of AXL may silence AXL and AXL

overexpression had been observed in some cancers367-371.

59

Sodium bisulfite cloning/sequencing enables the quantification of cytosine methylation at several

consecutive CpG sites373,374. The first step in sodium bisulfite cloning/sequencing is to treat the

genomic DNA with sodium bisulfite (under specific conditions) and then purify the resulting

DNA fragments. The second step is to amplify your sodium bisulfite-treated region of interest

using Polymerase Chain Reaction (PCR) amplification. The PCR amplification reaction

incorporates thymines instead of uracils into the daughter strands (recall how unmethylated

cytosines are converted by the sodium bisulfite treatment into uracils, sodium bisulfite treatment

does not alter methylated cytosines). The third step is to clone your PCR products into vectors.

The fourth step is to transform the E coli cells with the vector. The fifth step is to select E coli

colonies. The sixth step is to isolate and purify the plasmid DNA from the selected E coli

colonies. The seventh step is to sequence the plasmid DNA from the selected E coli colonies.


Healthcare Corporation), 10*BF (Qiagen), dNTPs (BioBasic), oligonucleotides (Integrated DNA

Technologies), HotStar Taq (Qiagen), and sodium bisulfite-treated DNA. The amounts and

concentrations of each of these reagents for the PCR amplification reactions are shown in

Appendices Table A-14. The thermal cycler programs for the PCR amplification reactions are

shown in Appendices Table A-15. The primers used for PCR amplification reactions are listed in

Appendices Table A-16.

The TOPO TA Cloning Kit manufactured by Invitrogen was used for PCR product cloning. The

GenElute Plasmid Miniprep Kit manufactured by Sigma was used to isolate and purify the

plasmid DNA.

2.14 Allelic Expression Analyses of AXL in Humans

I chose to carry out allelic expression analyses of AXL in humans since several candidate

paternally methylated CpG sites were identified in the promoter region of AXL, CpG methylation

in the promoter region of AXL may silence AXL, and AXL overexpression had been observed in

some cancers367-371.

60

Dr. Dalila Pinto from the Scherer laboratory located in the Genetics and Genome Biology

program at the Hospital for Sick Children collaborated on the project to provide genotype data

regarding AXL. These data were produced by the Illumina 1M-Duo v3.0 microarray for 90

persons from the Yoruba of Ibadan (YRI) and 90 persons of Northern and Western European

ancestry (CEU)375. Only one CEU individual had an AXL heterozygous transcribed SNP

(rs1051008). Thirty-three YRI individuals from 20 different pedigrees had the same AXL

heterozygous transcribed SNP (rs1051008). No other AXL heterozygous transcribed SNPs were

identified in either population. The Scherer laboratory provided me with DNA and RNA sets

from lymphoblastoid cell lines (LCLs) derived from blood of 4 informative heterozygous

individuals (these individuals were informative because it could be determined which parent

contributed which allele). cDNA was synthesized using SuperScript III reverse transcriptase

(Invitrogen) following the manufacturer’s instructions.

The genotypes for the 4 heterozygous individuals were validated using targeted pyrosequencing

(allelic quantification) by the PyroMark Q24 according to the manufacturer’s protocol. The

genomic region of interest (with SNP rs1051008) was amplified using PCR (polymerase chain

reaction). Biotin was not directly attached to the primers; instead an M13 sequence was attached

to the 5’ end of the primers, which annealed to a Biotin-M13 Universal Primer complex. The

PCR products were sequenced using pyrosequencing reactions. The pyrosequencing reaction

adds nucleotides one at a time in a designated order and the pyrosequencer measures the

proportion of nucleotides incorporated into the PCR products. A comparison of the proportion of

nucleotides incorporated provides the relative amount of each nucleotide in the DNA (we would

expect a 1:1 ratio for a heterozygous SNP).


Healthcare Corporation), 10*BF (Qiagen), magnesium chloride (Qiagen), dNTPs (BioBasic),

M13-Biotin Universal Primer (Integrated DNA Technologies), oligonucleotides (Integrated

DNA Technologies), HotStar Taq (Qiagen), and DNA. The amounts and concentrations of each

of these reagents for the PCR amplification reactions are shown in Appendices Table A-17. The

thermal cycler program for the PCR amplification reactions is shown in Appendices Table A-18.

The primers used for PCR amplification reactions are listed in Appendices Table A-19. The

primer used for pyrosequencing reactions is listed in Appendices Table A-20.

61

The double stranded cDNA was pyrosequenced (almost exactly like the procedure mentioned

above, except that double stranded cDNA is substituted for DNA) to determine relative allelic

expression (using the rs1051008 SNP). Only one out of the 4 informative heterozygous

individuals had preferential allelic expression of AXL. This individual’s parents’ genotypes were

retrieved from the genotype data of Dr. Dalila Pinto.

63

Chapter 3 Results

3 Results

I hypothesized that candidate parent-of-origin-specific differentially methylated CpG sites

(DMCpGs), some of which may reside in parent-of-origin-specific DMRs associated with nearby

known and/or novel imprinted genes in humans, could be identified by comparing the CpG

methylation of individual CpG sites in human uniparental and biparental tissues. To test this

hypothesis, sodium bisulfite-treated DNA from human uniparental tissues, androgenetic

complete hydatidiform mole (AnCHM) and mature cystic ovarian teratoma (MCT), and human

biparental tissues, blood and placenta, was hybridized to Illumina Infinium Human

Methylation27 promoter microarrays, which assess the proportion of CpG methylation at 27,578

individual CpG sites. The CpG methylation data for the 1092 sex chromosome CpG sites

represented on the Illumina Infinium Human Methylation27 promoter microarray was excluded.

Statistical values (used to characterize CpG methylation) were calculated for the remaining

26,486 autosomal CpG sites represented on the Illumina Infinium Human Methylation27

promoter microarray. Some of the statistical values (used to characterize CpG methylation)

specific to CpG sites located in known parent-of-origin-specific DMRs (associated with known

imprinted genes) were used to set thresholds/criteria to identify candidate parent-of-origin-

specific differentially methylated CpG sites (DMCpGs), some of which may reside in parent-of-

origin-specific DMRs associated with nearby known and/or novel imprinted genes in humans.

3.1 CpG Methylation in Known Parent-of-Origin-Specific DMRs Associated with Known Imprinted Genes

CpG sites represented on the Illumina Infinium Human Methylation27 promoter microarray

located in known parent-of-origin-specific DMRs (associated with known imprinted genes)

displayed differential CpG methylation in biparental tissues, blood and placenta, when compared

to uniparental tissues, AnCHM and MCT (Figure 3-1)359. This was expected as several studies

have noted differential CpG methylation in biparental tissues when compared to uniparental

64

tissues for CpG sites located in several known parent-of-origin-specific DMRs associated with

known imprinted genes163,164,166,176,376,377.

Since the Illumina Infinium Human Methylation27 promoter microarray does not target any CpG

sites within known intergenic parent-of-origin-specific DMRs (associated with known imprinted

genes), IC1 (also known as H19DMR) and IG-DMR, targeted quantitative sodium bisulfite

pyrosequencing was used to assess percent CpG methylation in blood, placenta, AnCHM, and

MCT. IC1 (H19DMR) displayed differential CpG methylation in blood and placenta, a gain of

CpG methylation in the paternal UPD11p15 lymphoblastoid cell line and AnCHMs, and a loss of

CpG methylation in the MCT (Figure 3-2A). IG-DMR presented differential CpG methylation in

blood and placenta, a gain of CpG methylation in the paternal UPD14 blood and AnCHMs, and a

loss of CpG methylation in the maternal UPD14 blood and MCT (Figure 3-2B).

For comparisons to the known paternally methylated intergenic DMRs (associated with known

imprinted genes), IC1 (H19DMR) and IG-DMR, targeted quantitative sodium bisulfite

pyrosequencing was used to assess percent CpG methylation in known maternally methylated

DMRs (associated with known imprinted genes) DIRAS3 DMR and IC2 (KvDMR1) in blood,

placenta, AnCHM, and MCT. The DIRAS3 DMR displayed differential CpG methylation in

blood and placenta, a loss of CpG methylation in the AnCHMs, and a gain of CpG methylation

in the MCT (Figure 3-2C). IC2 (KvDMR1) presented differential CpG methylation in blood and

placenta, a loss of CpG methylation in the paternal UPD11p15 lymphoblastoid cell line and

AnCHMs, and a gain of CpG methylation in the MCT (Figure 3-2D).

3.2 Candidate Parent-of-Origin-Specific Differentially Methylated CpG Sites (DMCpGs)

The criteria I used for candidate maternally methylated CpG sites in blood produced 365

candidates (Appendices Table A-6). The criteria I utilized for candidate maternally methylated

CpG sites in placenta produced 491 candidates (Appendices Table A-7). The original criteria for

candidate paternally methylated CpG sites in blood generated 0 candidates. The original criteria

for candidate paternally methylated CpG sites in placenta also produced produced 0 candidates.

Since both the original criteria for candidate paternally methylated CpG sites in blood and the

65

original criteria for candidate paternally methylated CpG sites in placenta were too strict

(allowed less variation) in comparison to the criteria for candidate maternally methylated CpG

sites in blood and the criteria for candidate maternally methylated CpG sites in placenta, the

criteria for candidate paternally methylated CpG sites in blood was loosened by adapting the

criteria for candidate maternally methylated CpG sites in blood and the criteria for candidate

paternally methylated CpG sites in placenta was loosened by adapting the criteria for candidate

maternally methylated CpG sites in placenta. The adapted criteria for candidate paternally

methylated CpG sites in blood generated 77 candidates (Appendices Table A-8). The adapted

criteria for candidate paternally methylated CpG sites in placenta produced 116 candidates

(Appendices Table A-9). There are 101 overlapping (i.e., present in both blood and placenta

lists) candidate maternally methylated CpG sites (Appendices Table A-21). There are 26

overlapping (i.e., present in both blood and placenta lists) candidate paternally methylated CpG

sites (Appendices Table A-22).

Interestingly, some overlapping (i.e., present in both blood and placenta lists) candidate parent-

of-origin-specific differentially methylated CpG sites (DMCpGs) had the same CpG methylation

pattern as known parent-of-origin-specific DMRs (associated with known imprinted genes) less

than a kilobase away (Appendices Table A-23). I postulated that these candidate parent-of-

origin-specific differentially methylated CpG sites (DMCpGs) represent components of these

known parent-of-origin-specific DMRs (associated with known imprinted genes).

Two candidate maternally methylated CpG sites were identified in the promoter region of the

known paternally expressed imprinted gene NAP1L5378, which at the time had no known

associated parent-of-origin-specific DMR. In an effort to determine if the genomic region in

between these two candidate maternally methylated CpG sites is maternally methylated, I carried

out quantitative sodium bisulfite pyrosequencing targeting a portion of the genomic region in

between these two candidate maternally methylated CpG sites in blood, placenta, AnCHM, and

MCT. The assessed CpG sites displayed differential CpG methylation in blood and placenta, a

loss of CpG methylation in the paternal UPD4 lymphoblastoid cell line and AnCHMs, and a gain

of CpG methylation in the MCT (Figure 3-3). These results suggest that the region may be

maternally methylated. Perhaps this region participates in regulating the imprinted gene

expression pattern of NAP1L5.

66

One candidate paternally methylated CpG site was identified in the promoter region of the

known maternally expressed imprinted gene ZNF597379, which at the time had no recognized

associated parent-of-origin-specific DMR. In an attempt to find out if the genomic region

upstream of the candidate paternally methylated CpG site is paternally methylated, quantitative

sodium bisulfite pyrosequencing targeting a segment of the genomic region upstream of the

candidate paternally methylated CpG site was performed in blood, placenta, AnCHM, and MCT.

The evaluated CpG sites displayed differential CpG methylation in blood and placenta, a gain of

CpG methylation in the AnCHMs, and a loss of CpG methylation in the MCT (Figure 3-4).

These results suggest that the genomic region upstream of the candidate paternally methylated

CpG site may be paternally methylated. Maybe this entire region is paternally methylated and

participates in regulating the imprinted gene expression pattern of ZNF597.

One candidate maternally methylated CpG site was detected in the promoter region of RIMS2.

This candidate maternally methylated CpG site had differential CpG methylation (~50% CpG

methylation) in blood and placenta (Figure 3-5A). In an endeavour to determine if the genomic

region upstream of the candidate maternally methylated CpG site is maternally methylated,

quantitative sodium bisulfite pyrosequencing targeting a section of the genomic region upstream

of the candidate paternally methylated CpG site was carried out in blood, placenta, AnCHM, and

MCT. Unexpectedly, the assessed CpG sites upstream of the candidate maternally methylated

CpG site displayed differential CpG methylation in the placenta and MCT, a gain of CpG

methylation in the blood, and a loss of CpG methylation in the AnCHMs (Figure 3-5B). These

results suggest that the genomic region upstream of the candidate maternally methylated CpG

site is not maternally methylated. The candidate maternally methylated CpG site may be a part of

a maternally methylated DMR that does not extend upstream. This maternally methylated DMR

might regulate the expression of RIMS2.

One candidate paternally methylated CpG site was detected in the promoter region of AXL; AXL

overexpression has been observed in some cancers, such as skin, lung, prostate, breast, and

gastric cancer367-371. This candidate paternally methylated CpG site had one of the highest CpG

methylation differences between the best AnCHM and MCT. In an effort to find out if the

genomic region surrounding the candidate paternally methylated CpG site is paternally

methylated, I performed quantitative sodium bisulfite pyrosequencing targeting the genomic

region surrounding the candidate paternally methylated CpG site in blood, placenta, AnCHM,

67

and MCT. The evaluated CpG sites displayed differential CpG methylation in blood and

placenta, a gain of CpG methylation in the AnCHMs, and a loss of CpG methylation in the MCT

(Figure 3-6). These results suggested that the genomic region surrounding the candidate

paternally methylated CpG site is paternally methylated. In an attempt to support and determine

the boundaries of the paternally methylated DMR (within the promoter region of AXL), sodium

bisulfite cloning/sequencing targeting the promoter region of AXL was performed in the MCT,

one AnCHM, and one placenta. Using targeted sodium bisulfite cloning/sequencing, the

boundaries of the paternally methylated DMR and one other candidate paternally methylated

CpG site were identified (Figure 3-7). Perhaps these candidate paternally methylated CpG sites

are part of a paternally methylated DMR that regulates the expression of AXL.

3.3 Allelic Expression of AXL in Humans Since several candidate paternally methylated CpG sites were identified in the promoter region

of AXL, CpG methylation in the promoter region of AXL may silence AXL, and AXL

overexpression had been observed in some cancers367-371, allelic expression analyses of AXL (in

humans) were performed. The genotypes of four informative individuals heterozygous for AXL

(SNP rs1051008) were validated using targeted pyrosequencing (allelic quantification) using

their lymphoblastoid cell line (LCL) DNA (Table 3-1). Relative allelic expression was estimated

in these individuals using targeted pyrosequencing (allelic quantification) on cDNA generated

from their lymphoblastoid cell line (LCL) RNA (Table 3-1). Although AXL was preferentially

expressed from the maternal chromosome in one informative heterozygous individual, AXL was

not expressed preferentially from any parental chromosome in the other three informative

heterozygous individuals (Table 3-1). These results suggest AXL may have polymorphic

imprinting in human blood.

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Figure 3-1: CpG Methylation in DMRs Associated With Known Imprinted Genes

Heatmap displaying the CpG methylated proportions of Illumina Infinium Human Methylation27 promoter

microarray CpG sites located in known parent-of-origin-specific DMRs associated with known imprinted genes.

Each column represents one sample run on the Illumina Infinium Human Methylation27 promoter microarray. Each

row represents one unique CpG site (labeled with its unique Illumina ID and the gene promoter it is located in)

targeted on the Illumina Infinium Human Methylation27 promoter microarray that is located within a known parent-

of-origin-specific DMR associated with a known imprinted gene. The two CpG sites at the bottom are paternally

methylated (these two CpG sites are located in the known paternally methylated GNAS NESP55 DMR associated

with known imprinted gene GNAS); the other CpG sites are maternally methylated. The number of microarray CpG

sites within each known parent-of-origin-specific DMR (associated with known imprinted genes) varies. The

samples are labeled WBC (white blood cell), placenta, AnCHM (androgenetic complete hydatidiform mole), and

MCT (mature cystic ovarian teratoma). Illumina Infinium Human Methylation27 promoter microarray CpG sites on

the list of CpG sites located in known parent-of-origin-specific DMRs (associated with known imprinted genes)

displayed differential CpG methylation in biparental tissues, blood and placenta, when compared to uniparental

tissues, AnCHM and MCT. Adapted from Genome Research, Vol. 21 (Issue 3), Choufani et al., A novel approach

identifies new differentially methylated regions (DMRs) associated with imprinted genes, 465-476, Copyright

(2011), with permission from Cold Spring Harbor Laboratory Press.

69

Figure 3-1: CpG Methylation in DMRs Associated With Known Imprinted Genes

70

Figure 3-2: CpG Methylation within Four DMRs Associated with Imprinted Genes

Box-and-whisker plot showing the distribution of sodium bisulfite pyrosequencing percent CpG methylation for: A)

3 consecutive CpG sites within the paternally methylated IC1 (H19DMR) on human chromosome 11 for each tissue.

The data came form 15 blood, 10 placenta, 3 AnCHM, 1 paternal UPD11p15 lymphoblastoid cell line, and 1 MCT

sample; B) 5 consecutive CpG sites within the paternally methylated IG-DMR on human chromosome 14 for each

tissue. The data came from 17 blood, 9 placenta, 3 AnCHM, 1 paternal UPD14 blood, 1 MCT, and 1 maternal

UPD14 blood sample; C) 3 consecutive CpG sites within the maternally methylated DIRAS3 DMR on human

chromosome 1 for each tissue. The data came from 16 blood, 5 placenta, 3 AnCHM, and 1 MCT sample; D) 5

consecutive CpG sites within the maternally methylated KvDMR1 on human chromosome 11 for each tissue. The

data came from 15 blood, 10 placenta, 3 AnCHM, 1 paternal UPD11p15 lymphoblastoid cell line, and 1 MCT

sample. These results suggest known parent-of-origin-specific DMRs (associated with known imprinted genes) are

differentially methylated in biparental tissues, blood and placenta, when compared to uniparental tissues, AnCHM

and MCT.

71

A

B


72

C

D


(Continued)

73

Figure 3-3: CpG Methylation within the Candidate NAP1L5 DMR

Box-and-whisker plot showing the distribution of: A) Illumina Infinium Human Methylation27 promoter microarray

percent CpG methylation for NAP1L5 CpG sites cg01026744 and cg12759554 for each tissue. The data came from

16 blood, 5 placenta, 3 AnCHM, 1 paternal UPD4 lymphoblastoid cell line, and 1 MCT sample; B) Sodium bisulfite

pyrosequencing percent CpG methylation for 6 consecutive CpG sites within the candidate maternally methylated

NAP1L5 DMR for each tissue. The data came from 15 blood, 10 placenta, 3 AnCHM, 1 paternal UPD4

lymphoblastoid cell line, and 1 MCT sample. Genomic regions within the candidate maternally methylated DMR for

known paternally expressed imprinted gene NAP1L5 display differential CpG methylation in blood and placenta, a

loss of CpG methylation in the paternal UPD4 lymphoblastoid cell line and AnCHMs, and a gain of CpG

methylation in the MCT.

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A

B

Figure 3-3: CpG Methylation within the Candidate NAP1L5 DMR

75

Figure 3-4: CpG Methylation within the Candidate ZNF597 DMR


percent CpG methylation for ZNF597 CpG site cg14654875 for each tissue. The data came from 16 blood, 5

placenta, 3 AnCHM, and 1 MCT sample. Adapted from Genome Research, Vol. 21 (Issue 3), Choufani et al., A

novel approach identifies new differentially methylated regions (DMRs) associated with imprinted genes, 465-476,

Copyright (2011), with permission from Cold Spring Harbor Laboratory Press; B) Sodium bisulfite pyrosequencing

percent CpG methylation for 4 consecutive CpG sites within the candidate paternally methylated ZNF597 DMR for

each tissue. The data came from 14 blood, 4 placenta, 1 AnCHM, and 1 MCT sample. Genomic regions within the

candidate paternally methylated DMR for known maternally expressed imprinted gene ZNF597 display differential

CpG methylation in blood and placenta, a gain of CpG methylation in the AnCHMs, and a loss of CpG methylation

in the MCT.

76

A

B

Figure 3-4: CpG Methylation within the Candidate ZNF597 DMR

77

Figure 3-5: CpG Methylation within the Candidate RIMS2 DMR


percent CpG methylation for RIMS2 CpG site cg05341878 for each tissue. The data came from 16 blood, 5 placenta,

3 AnCHM, and 1 MCT sample; B) Sodium bisulfite pyrosequencing percent CpG methylation for 2 consecutive

CpG sites approximately 100 bases upstream of the candidate maternally methylated RIMS2 CpG site for each

tissue. The data came from 17 blood, 12 placenta, 3 AnCHM, and 1 MCT sample. The RIMS2 CpG site cg05341878

displays differential CpG methylation in blood and placenta, a loss of CpG methylation in the AnCHMs, and

differential CpG methylation in the MCT. A genomic region approximately 100 bases upstream of the RIMS2 CpG

site cg05341878 displays differential CpG methylation in the placenta and MCT, a gain of CpG methylation in the

blood, and a loss of CpG methylation in the AnCHMs.

78

A

B

Figure 3-5: CpG Methylation within the Candidate RIMS2 DMR

79

Figure 3-6: CpG Methylation within the Candidate AXL DMR


percent CpG methylation for AXL CpG site cg14892768 for each tissue. The data came from 16 blood, 5 placenta, 3

AnCHM, and 1 MCT sample; B) Sodium bisulfite pyrosequencing percent CpG methylation for 4 consecutive CpG

sites within the candidate paternally methylated AXL DMR for each tissue. The first CpG site analyzed is AXL CpG

site cg14892768 on the Illumina Infinuium Human Methylation27 promoter microarray. The data came from 15

blood, 10 placenta, 3 AnCHM, and 1 MCT sample. Adapted from Genome Research, Vol. 21 (Issue 3), Choufani et

al., A novel approach identifies new differentially methylated regions (DMRs) associated with imprinted genes, 465-

476, Copyright (2011), with permission from Cold Spring Harbor Laboratory Press. A genomic region within the

candidate paternally methylated DMR for candidate imprinted gene AXL displays differential CpG methylation in

blood and placenta, a gain of CpG methylation in the AnCHMs, and a loss of CpG methylation in the MCT.

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A

B

Figure 3-6: CpG Methylation within the Candidate AXL DMR

81

Figure 3-7: Candidate AXL DMR Sodium Bisulfite Sequencing

CpG methylation of the candidate paternally methylated AXL DMR. A) Genomic location of the human AXL

promoter with the 355 base-pair region analyzed for CpG methylation (using sodium bisulfite cloning/sequencing)

indicated by a red filled rectangle. The Illumina Infinium Human Methylation27 promoter microarray AXL CpG site

cg14892768 is located within this red rectangle. Rakyan et al. (2008)380 performed CpG methylation analyses using

MeDIP on sperm and placenta and their CpG methylation data for the AXL promoter region has been integrated into

the Ensembl Genome Browser screenshot (shown in the MeDIP-chip Placenta and MeDIP-chip Sperm rows, chip in

this case refers to microarrays). The green region within the MeDIP-chip Placenta row is intermediately methylated

(~50% CpG methylation). The blue region within the MeDIP-chip Sperm row is methylated (~100% CpG

methylation); B) DNA sequence of the 355 base-pair genomic region that had its CpG methylation analyzed using

sodium bisulfite cloning/sequencing. The CpG dinucleotides are in bold. The underlined (and #ed) CpG site is the

Illumina Infinium Human Methylation27 promoter microarray AXL CpG site cg14892768. The # CpG sites had their

CpG methylation analyzed using targeted quantitative sodium bisulfite pyrosequencing (Figure 3-6B); C) Sodium

bisulfite cloning/sequencing results for the 14 CpG sites within the 355 base-pair fragment from the AXL promoter

region in the MCT (on the right), one AnCHM (on the left), and one placenta sample (in the middle). Each row

corresponds to a specific clone. Each column corresponds to a specific CpG site (the CpG sites are ordered by their

genomic location (p-arm to q-arm, left to right)). A black filled circle represents a methylated CpG site. A white

filled circle represents an unmethylated CpG site. The Illumina Infinium Human Methylation27 promoter

microarray AXL CpG site cg14892768 is indicated by the black arrows at the bottom. Beneath each set of results is

the tissue type (e.g., MCT). A genomic region within the candidate paternally methylated DMR for candidate

imprinted gene AXL displays differential CpG methylation in one placenta, a gain of CpG methylation in one

AnCHM, and a loss of CpG methylation in the MCT. Not even one genomic single nucleotide variant was observed

in the sodium bisulfite-treated DNA. Adapted from Genome Research, Vol. 21 (Issue 3), Choufani et al., A novel

approach identifies new differentially methylated regions (DMRs) associated with imprinted genes, 465-476,

Copyright (2011), with permission from Cold Spring Harbor Laboratory Press.

82

A

B AGGCAGGGGTGCTGAGAAGGCGGCTGCTGGGCAGAGCCGGTGGCAAGGGCCTCCCCTGCCGCTGTGCCAGGCAG

GCAGTGCCAAATCCGGGGAGCCTGGAGCTGGGGGGAGGGCCGGGGACAGCCCGGCCCTGCCCCCTCCCCCGCTG

GGAGCCCAACAACTTCTGAGGAAAGTTTGGCACCCATGGCGTGGCGGTGCCCCAGGATGGGCAGGGTCCCG#CTG

GCCTGGTGCTTGGCG#CTGTGCG#GCTGGGCG#TGCATGGCCCCCAGGGGTGAGTGATGGGGGCTCCTTGGGGCAG

GGATCCCCTCGGAGGGGCTGGGGCAGGGGTAGGAGGTGGGGGATGATGGCAGGTGTGGGG

C

Figure 3-7: Candidate AXL DMR Sodium Bisulfite Sequencing

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Table 3-1: AXL SNP Quantification in DNA and RNA of Informative Individuals

The AXL SNP is rs1051008. The SNP quantification was performed using targeted pyrosequencing (allelic

quantification). The DNA and RNA were extracted from lymphoblastoid cell lines (LCLs). The informative

individuals mentioned in the title are informative because they are heterozygous for the AXL SNP rs1051008 and in

these individuals it can be determined which of their parents contributed each allele present. The genotype of the

father of individual 35326 is GG at the AXL SNP rs1051008 (determined from the Hapmap data generated on the

Illumina 1M-Duo v3). The genotype of the mother of individual 35326 is AA at the AXL SNP rs1051008

(determined from the Hapmap data generated on the Illumina 1M-Duo v3). In one informative heterozygous

individual (shaded in light blue), AXL was preferentially expressed from the maternal chromosome. In the other

three heterozygous individuals, AXL was not expressed preferentially from any parental chromosome. Adapted from

Genome Research, Vol. 21 (Issue 3), Choufani et al., A novel approach identifies new differentially methylated

regions (DMRs) associated with imprinted genes, 465-476, Copyright (2011), with permission from Cold Spring

Harbor Laboratory Press.

Individual ID DNA RNA

34969 A: 55%; G: 45% A: 59%; G:41%

35158 A: 57%; G: 43% A: 53%; G: 47%

35167 A: 58%; G: 42% A: 56%; G: 44%

35326 A: 57%; G: 43% A: 82%; G: 18%

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Chapter 4 Discussion

4 Discussion

Various approaches have been utilized to identify novel imprinted genes and their associated

parent-of-origin-specific differentially methylated regions (DMRs) in humans335-341. I developed

a new approach to identify candidate parent-of-origin-specific differentially methylated regions

(DMRs), some of which may be associated with nearby known and/or novel imprinted genes in

humans. Sodium bisulfite-treated DNA from human uniparental tissues, androgenetic complete

hydatidiform mole (AnCHM) and mature cystic ovarian teratoma (MCT), and human biparental

tissues, blood and placenta, was hybridized to CpG methylation microarrays to identify candidate

parent-of-origin-specific differentially methylated CpG sites (DMCpGs), some of which may

reside in parent-of-origin-specific differentially methylated regions (DMRs) associated with

nearby known and/or novel imprinted genes.

4.1 More Complete Picture of CpG Methylation in Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes)

I assayed CpG methylation within fourteen known parent-of-origin-specific DMRs (associated

with known imprinted genes) in blood, placenta, AnCHM, and MCT (Figure 3-1; Figure 3-2).

Many studies have assessed the CpG methylation state of known parent-of-origin-specific DMRs

(associated with known imprinted genes) in biparental tissues. However, very few studies have

examined the CpG methylation state of known parent-of-origin-specific DMRs (associated with

known imprinted genes) in uniparental tissues, AnCHM and MCT (Appendices Table A-1).

Notably, many known parent-of-origin-specific DMRs (associated with known imprinted genes)

have not had their CpG methylation investigated in AnCHMs and/or MCTs in previous studies

(Appendices Table A-1). In biparental and uniparental tissues, I have examined the CpG

methylation within many of these known parent-of-origin-specific DMRs associated with known

imprinted genes (Figure 3-1; Figure 3-2).

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By using the Illumina Infinium Human Methylation27 promoter microarray to assess the CpG

methylation of CpG sites located in additional known parent-of-origin-specific DMRs

(associated with known imprinted genes) in uniparental and biparental tissues, a more complete

picture of CpG methylation within known parent-of-origin-specific DMRs (associated with

known imprinted genes) was produced. The extra data increased the number of known and

candidate parent-of-origin-specific differentially methylated CpG sites (DMCpGs) identified by

my method. Some of the additional candidate parent-of-origin-specific DMCpGs may reside

within parent-of-origin-specific DMRs associated with nearby known and/or novel imprinted

genes.

4.2 Biparental Tissues (When Compared to Uniparental Tissues) Have Differential CpG Methylation in Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes)

As expected, known parent-of-origin-specific DMRs (associated with known imprinted genes)

were differentially methylated in biparental tissues, blood and placenta, when compared to

uniparental tissues, AnCHM and MCT (Figure 3-1; Figure 3-2). A few previous studies have

contrasted the percent CpG methylation of some known parent-of-origin-specific DMRs

(associated with known imprinted genes) in biparental and uniparental tissues163,166,176. In the

known paternally methylated IC1 (H19DMR), Miura et al. (1999)176 reported hypomethylation

in MCTs (when compared to blood) and El-Maarri et al. (2003)166 reported hypermethylation in

AnCHMs (when compared to blood). In the known maternally methylated SNRPN DMR, Miura

et al. (1999)176 noted hypermethylation in MCTs (when compared to blood) and El-Maarri et al.

(2003)166 noted hypomethylation in AnCHMs (when compared to blood).

Due to the differences in CpG methylation in known parent-of-origin-specific DMRs (associated

with known imprinted genes) between androgenetic complete hydatidiform moles (AnCHMs),

mature cystic ovarian teratoma (MCT), and biparental tissues, I was able to identify candidate

parent-of-origin-specific differentially methylated CpG sites (DMCpGs) using my approach.

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4.3 Variable CpG Methylation in Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes)

Interestingly, the variability in the CpG methylation of some known parent-of-origin-specific

DMRs (associated with known imprinted genes) varied between tissues (Figure 3-1; Figure 3-2).

Results from several previous studies have also suggested that the variability in the CpG

methylation of some known parent-of-origin-specific DMRs (associated with known imprinted

genes) varies between tissues166,175,381. Perhaps these known parent-of-origin-specific DMRs

(associated with known imprinted genes) have different CpG methylation natural histories in

different cell types (DNA in blood generally comes from neutrophils, lymphocytes, monocytes,

and eosinophils382; DNA in placenta generally comes from cytotrophoblasts,

syncytiotrophoblasts, and fibroblasts383,384). Such differences in CpG methylation could be due to

differences in CpG methylation maintenance385-389, de novo CpG methylation385,390,391, and/or

other epigenetic processes392-395.

Differences in cell type composition of the individual tissue samples may also factor into the

difference in variability in the CpG methylation of some known parent-of-origin-specific DMRs

(associated with known imprinted genes) in different tissues381. Some biparental cell types may

not contain parent-of-origin-specific CpG methylation at some parent-of-origin-specific

DMRs396. The absence of parent-of-origin-specific CpG methylation at some parent-of-origin-

specific DMRs in some biparental cell types may be more common at secondary parent-of-

origin-specific DMRs when compared to primary parent-of-origin-specific DMRs. The

epigenetic processes required to establish parent-of-origin-specific CpG methylation at some

secondary parent-of-origin-specific DMRs in some biparental cell types may be perturbed.

Notably, some uniparental cell types (in AnCHM or MCT) may not contain completely

methylated/unmethylated genomic regions within some parent-of-origin-specific DMRs. This

circumstance may be more common at secondary parent-of-origin-specific DMRs (when

compared to primary parent-of-origin-specific DMRs). The epigenetic processes required to

methylate/unmethylate the secondary parent-of-origin-specific DMR in some uniparental cell

types may be disrupted.

Since some biparental cell types, such as neutrophils in blood or cytotrophoblasts in placenta,

may not contain parent-of-origin-specific CpG methylation at some parent-of-origin-specific

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DMRs, candidate maternally methylated CpG sites and candidate paternally methylated CpG

sites were determined for blood and placenta separately.

4.4 Poor Microarray Coverage of Known Paternally Methylated DMRs

It was not surprising that the Illumina Infinium Human Methylation27 promoter microarray had

very poor coverage of known paternally methylated DMRs (when compared to the coverage of

known maternally methylated DMRs) (Appendices Table A-5). There are 5 known paternally

methylated DMRs in the human genome16,167,172,175,181,190,198,397,398. Of the five known paternally

methylated DMRs, two are located within intergenic regions, two are located within gene

promoters, and one is located in the gene body of IGF2. For comparison, there are 14 known

maternally methylated DMRs in the human genome10,18,19,24,26,28,163,164,170,177,180,188,196,227,346,399-403.

Of the 14 known maternally methylated DMRs, 12 are located within gene promoters, one is

located in the gene body of GRB10, and the last one is located in the gene body of IGF2R.

The poor microarray coverage of known paternally methylated DMRs may have caused the

original criteria for candidate paternally methylated CpG sites in blood and candidate paternally

methylated CpG sites in placenta to be more strengthened than the criteria for candidate

maternally methylated CpG sites in blood and candidate maternally methylated CpG sites in

placenta. Therefore, the criteria for candidate paternally methylated CpG sites in blood were

loosened by adapting the criteria for candidate maternally methylated CpG sites in blood. The

criteria for candidate paternally methylated CpG sites in placenta were loosened by adapting the

criteria for candidate maternally methylated CpG sites in placenta. This decreased the bias

against paternally methylated CpG sites.

Considering the distribution of known paternally methylated DMRs (associated with known

imprinted genes) and known maternally methylated DMRs (associated with known imprinted

genes) in the human genome, it was expected that there would be more candidate maternally

methylated CpG sites than candidate paternally methylated CpG sites (Table A-6; Table A-7;

Table A-8; Table A-9).

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4.5 Very Few Candidate Imprinted Loci Identified By More Than One Study

My method identified 365 candidate maternally methylated CpG sites in blood (Table A-6), 491

candidate maternally methylated CpG sites in placenta (Table A-7), 77 candidate paternally

methylated CpG sites in blood (Table A-8), and 116 candidate paternally methylated CpG sites

in placenta (Table A-9). Three hundred sixty four candidate maternally methylated CpG sites in

blood, 488 candidate maternally methylated CpG sites in placenta, 77 candidate paternally

methylated CpG sites in blood, and 116 candidate paternally methylated CpG sites in placenta

are associated with approximately 340, 440, 70, and 100 genes respectively. One hundred

overlapping (i.e., present in both blood and placenta lists) candidate maternally methylated CpG

sites are associated with ~90 genes. Twenty six overlapping (i.e., present in both blood and

placenta lists) candidate paternally methylated CpG sites are associated with ~20 genes.

One candidate maternally methylated CpG site in blood is associated with candidate imprinted

gene TCEB3C, which was identified by Strichman-Almashanu et al. (2002)335 using CpG

methylation sensitive and CpG methylation insensitive restriction enzymes on genomic DNA

from two uniparental tissues (AnCHM (androgenetic complete hydatidiform mole) and MCT

(mature cystic ovarian teratoma)), followed by cloning and sequencing.

One candidate maternally methylated CpG site in blood is associated with candidate imprinted

gene CDH18. One candidate maternally methylated CpG site in placenta is associated with

candidate imprinted gene FAM50B. One candidate paternally methylated CpG site in placenta is

associated with candidate imprinted gene PAOX. Five candidate paternally methylated CpG sites

in placenta are associated with candidate imprinted gene RBP5. One candidate paternally

methylated CpG site in placenta is associated with candidate imprinted gene HOXC4. One

candidate maternally methylated CpG site in blood is associated with candidate imprinted gene

PPAP2C. Candidate imprinted genes CDH18, FAM50B, PAOX, RBP5, HOXC4, and PPAP2C

were identified by Luedi et al. (2007)336 using computer algorithms to search the genome for

genes with DNA sequence features around known imprinted genes.

One candidate maternally methylated CpG site in placenta is associated with candidate imprinted

gene ATF5. One candidate maternally methylated CpG site in blood is associated with candidate

imprinted gene SLC7A7. One candidate maternally methylated CpG site in blood is associated

90

with candidate imprinted gene BCL2L14. One candidate maternally methylated CpG site in

placenta is associated with candidate imprinted gene TSPAN4. One candidate paternally

methylated CpG site in blood is associated with candidate imprinted gene BTN3A2. Candidate

imprinted genes ATF5, SLC7A7, BCL2L14, TSPAN4, and BTN3A2 were identified by Pollard et

al. (2008)337 using SNP microarrays to search for genes with differential allelic expression in

peripheral blood leukocytes (PBLs).

One candidate maternally methylated CpG site in placenta is associated with candidate imprinted

gene CUBN. One overlapping (i.e., present in both blood and placenta lists) candidate maternally

methylated CpG site is associated with candidate imprinted gene GRIN2B. One candidate

maternally methylated CpG site in blood is associated with candidate imprinted gene LIFR. One

candidate maternally methylated CpG site in placenta is associated with candidate imprinted

gene SLC10A2. One candidate maternally methylated CpG site in blood is associated with

candidate imprinted gene SLC4A4. One overlapping (i.e., present in both blood and placenta

lists) candidate paternally methylated CpG site is associated with known imprinted gene

ZNF597. Known imprinted gene ZNF597 and candidate imprinted genes CUBN, GRIN2B, LIFR,

SLC10A2, and SLC4A4, were identified by Maynard et al. (2008)338 by searching for genes with

RNA polymerase bound to only one of their two alleles.

The candidate maternally methylated CpG sites in placenta associated with candidate imprinted

genes TRPC3 and FAM50B were identified as candidate maternally methylated CpG sites by

Nakabayashi et al. (2011)340 using CpG methylation profiling of biparental tissues and blood

tissues with cells containing genome-wide maternal/paternal uniparental disomies. The candidate

maternally methylated CpG site in blood associated with candidate imprinted gene ZBTB16 was

identified as a candidate maternally methylated CpG site by Nakabayashi et al. (2011)340. The


site associated with known imprinted gene ZNF597 was identified as a candidate paternally

methylated CpG site by Nakabayashi et al. (2011)340.

The candidate paternally methylated CpG sites in placenta associated with candidate imprinted

genes AKAP10, PARP12, ACPL2, and PEX5 were identified as candidate paternally methylated

CpG sites by Yuen et al. (2011)341 using CpG methylation profiling of biparental diploid (one

maternal genome and one paternal genome) placentas, diandric triploid (two paternal genomes

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and one maternal genome) placentas, digynic triploid (2 maternal genomes and 1 paternal

genome) placentas, and androgenetic complete hydatidiform moles (AnCHMs). The candidate

maternally methylated CpG site in placenta associated with candidate imprinted gene FAM50B

was identified as a candidate maternally methylated CpG site by Yuen et al. (2011)341. The


site associated with candidate imprinted gene LEP was identified as a candidate paternally

methylated CpG site by Yuen et al. (2011)341.

Interestingly, candidate imprinted gene FAM50B was identified by Luedi et al. (2007)336,

Nakabayashi et al. (2011)340, Yuen et al. (2011)341, and I. Nakabayashi et al. (2011)340 and I have

identified the candidate paternally methylated CpG site associated with known imprinted gene

ZNF597.

4.6 Boundaries of Known Parent-of-Origin-Specific DMRs (Associated with Known Imprinted Genes) are Unknown

Candidate parent-of-origin-specific DMCpGs with the same methylation pattern as known

parent-of-origin-specific DMRs (associated with known imprinted genes) less than a kilobase

away were identified (Appendices Table A-23). Perhaps these candidate parent-of-origin-specific

DMCpGs are components of these known parent-of-origin-specific DMRs (associated with


Although many studies have added to what is known about the CpG methylation state within

known parent-of-origin-specific DMRs (associated with known imprinted genes) in different

tissues, the boundaries of known parent-of-origin-specific DMRs (associated with known

imprinted genes) are still unknown. Notably, there are studies that have crudely estimated the

boundaries of known parent-of-origin-specific DMRs (associated with known imprinted

genes)172,205.

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4.7 Candidate Maternally Methylated DMR in Promoter of Imprinted Gene NAP1L5

In the promoter region of known paternally expressed imprinted gene NAP1L5378, eight

candidate maternally methylated CpG sites were identified. Two of these eight candidate

maternally methylated CpG sites were identified using the Illumina Infinium Human

Methylation27 promoter microarray. The six other candidate maternally methylated CpG sites

were identified using sodium bisulfite pyrosequencing (Figure 3-3B). The eight candidate

maternally methylated CpG sites may be located within a maternally methylated DMR that

participates in regulating the imprinted gene expression pattern of NAP1L5. In mice, a

maternally methylated DMR associated with the known paternally expressed imprinted gene

Nap1l5 has been discovered in the promoter region of Nap1l5404. Perhaps maternal DNA

methylation in the promoter region of NAP1L5 silences the maternal NAP1L5 in tissue(s) where

NAP1L5 is imprinted.

4.8 Candidate Paternally Methylated DMR in Promoter of Imprinted Gene ZNF597

In the promoter region of known maternally expressed imprinted gene ZNF597379, five candidate

paternally methylated CpG sites were identified. One of these five candidate paternally

methylated CpG sites was identified using the Illumina Infinium Human Methylation27

promoter microarray (Figure 3-4A). The four other candidate paternally methylated CpG sites

were identified using sodium bisulfite pyrosequencing (Figure 3-4B). The five candidate

paternally methylated CpG sites may be located within a paternally methylated DMR that

participates in regulating the imprinted gene expression pattern of ZNF597. Maybe paternal

DNA methylation in the promoter region of ZNF597 silences the paternal ZNF597 in tissue(s)

where ZNF597 is imprinted.

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4.9 Candidate Maternally Methylated DMR in Promoter of RIMS2

In the promoter region of candidate imprinted gene RIMS2, one candidate maternally methylated

CpG site was identified using the Illumina Infinium Human Methylation27 promoter microarray

(Figure 3-5A). This candidate maternally methylated CpG site may be located within a

maternally methylated DMR that regulates the expression of RIMS2. Two CpG sites upstream of

the aforementioned candidate maternally methylated CpG site did not appear to be differentially

methylated depending on their parent-of-origin (Figure 3-5B). Perhaps the candidate maternally

methylated DMR does not extend to these two upstream CpG sites. Maybe maternal DNA

methylation in the region of the candidate maternally methylated CpG site silences the maternal

RIMS2 in some tissues.

4.10 Candidate Paternally Methylated DMR in Promoter of AXL

In the promoter region of candidate imprinted gene AXL, five candidate paternally methylated

CpG sites were identified. One candidate paternally methylated CpG site was identified using the

Illumina Infinium Human Methylation27 promoter microarray (Figure 3-6A). Three other

candidate paternally methylated CpG sites were identified using sodium bisulfite pyrosequencing

(Figure 3-6B). The last candidate paternally methylated CpG sites was identified using sodium

bisulfite cloning/sequencing (Figure 3-7). The five candidate paternally methylated CpG sites

may be located within a paternally methylated DMR that regulates the expression of AXL.

Plausibly, paternal DNA methylation in the promoter region of AXL silences the paternal AXL in

some tissues.

4.11 Polymorphic Imprinting of AXL

AXL was observed to have preferential parental expression in only one lymphoblastoid cell line

(one lymphoblastoid cell line (LCL) was established from each informative heterozygous

individual) (Table 3-1). Perhaps AXL, like paternally expressed imprinted gene IGF2405, has

polymorphic imprinting in human blood. AXL may have (polymorphic) imprinting in other

94

tissues or cell types and maybe at different developmental stages. Maybe the informative

heterozygous individual with preferential maternal expression of AXL in his/her lymphoblastoid

cell line (LCL) has preferential maternal expression of AXL in some of his/her other tissues

(possibly at different developmental stages). Other genes with polymorphic imprinting in

humans include IGF2R and SLC22A2406.

Interestingly, Axl is expressed preferentially from the maternal chromosome in the early mouse

conceptus359. Although the mouse conceptus usually loses its preferential parental expression of

Axl in later stages of development, sometimes preferential maternal expression of Axl is retained,

which is intriguing because polymorphic imprinting has not been described in mice. Perhaps

AXL is expressed preferentially from the maternal chromosome in the early human conceptus.

Maybe the human conceptus usually loses its preferential parental expression of AXL in later

stages of development, but sometimes retains preferential maternal expression of AXL, resulting

in polymorphic imprinting.

4.12 General Discussion

My hypothesis was that candidate parent-of-origin-specific DMCpGs, some of which may reside

in parent-of-origin-specific DMRs associated with nearby known and/or novel imprinted genes

in humans, can be identified by comparing the CpG methylation of individual CpG sites in

human uniparental and biparental tissues. Interestingly, some candidate parent-of-origin-specific

DMCpGs I identified were associated with known imprinted genes (NAP1L5 and ZNF597)

without known parent-of-origin-specific DMRs at the time; perhaps these candidate parent-of-

origin-specific DMCpGs are a part of the parent-of-origin-specific DMRs that regulate the

imprinting of these known imprinted genes. I also identified candidate parent-of-origin-specific

DMCpGs in the promoter region of AXL and allelic expression analyses of AXL in human

lymphoblastoid cell lines (LCLs) were performed. I discovered one lymphoblastoid cell line

(LCL) with preferential parental expression of AXL, which suggests AXL may have polymorphic

imprinting in human blood.

My approach to identify candidate imprinted genes was limited in that it identifies candidate

parent-of-origin-specific DMCpGs, not candidate imprinted genes. The genes around my

95

candidate parent-of-origin-specific DMCpGs may be imprinted. My approach was also limited in

that it could not be used to identify candidate parent-of-origin-specific DMCpGs genome-wide

because it utilized the Illumina Infinium Human Methylation27 microarray, which is a promoter

microarray that interrogates on average only two CpG sites in every gene promoter.

Unfortunately, the Illumina Infinium Human Methylation27 promoter microarray could not be

used to define the boundaries of DMRs. To define the boundary of a DMR, the CpG methylation

of the entire DMR, plus the surrounding of the DMR, would need to be defined.

4.13 Future Directions

I have identified candidate parent-of-origin-specific DMCpGs, which reside in candidate parent-

of-origin-specific DMRs, some of which may be associated with nearby novel imprinted genes in

humans. A future study could attempt to determine if genes nearby my candidate parent-of-

origin-specific DMCpGs are imprinted in humans using allelic expression analyses in different

tissues at different developmental stages. If any novel imprinted genes in humans are identified,

their homologues in other mammals, such as mice, could be checked for imprinting.

My approach could not be used to identify candidate DMCpGs genome-wide because it utilized

the Illumina Infinium Human Methylation27 promoter microarray, which interrogates on

average only two CpG sites in every gene promoter. A future study could replicate my study, but

instead of using the Illumina Infinium Human Methylation27 promoter microarray, use the 450K

Illumina Methylation BeadChip microarray. The 450K Illumina Methylation BeadChip

microarray would interrogate the methylation status of greater than 450,000 cytosine sites

covering CpG islands, CpG island shores, non-CpG methylated cytosines identified in human

stem cells, and miRNA promoter regions. Also, another future study could expand the scope of

my study by using next generation sodium bisulfite sequencing instead of microarray

technology. The next generation sodium bisulfite sequencing approach would allow one to assess

the methylation of significantly more cytosines in the human genome.

The boundaries for known parent-of-origin-specific DMRs (associated with known imprinted

genes) have yet to be determined in humans. Also, boundaries for my candidate parent-of-origin-

specific DMRs that may be associated with known human imprinted genes, NAP1L5 and

96

ZNF597, are unknown. A future study could determine the boundaries for these human DMRs,

perhaps using next generation sodium bisulfite sequencing.

4.14 Conclusions

Several approaches have been utilized to identify novel imprinted genes in humans. I developed

a new method to identify candidate imprinted genes in humans using the fact that imprinted

genes are often associated with nearby parent-of-origin-specific DMRs. I utilized sodium

bisulfite-based CpG methylation profiling of uniparental tissues, AnCHM and MCT, and

biparental tissues, blood and placenta, to identify candidate parent-of-origin-specific DMRs. I

identified candidate parent-of-origin-specific DMRs in the promoter regions of the known

imprinted genes NAP1L5 and ZNF597, which did not have known associated parent-of-origin-

specific DMRs at the time. I also identified a candidate paternally methylated DMR in the

promoter region of AXL and allelic expression analyses of AXL in human lymphoblastoid cell

lines (LCLs) were performed. In only one lymphoblastoid cell line (LCL), I discovered

preferential parental expression of AXL, which suggests AXL may have polymorphic imprinting

in human blood. Perhaps other candidate parent-of-origin-specific DMRs I identified are

associated with nearby novel imprinted genes not yet discovered. Maybe some of these novel

imprinted genes could help explain some unexplained cases of deregulated development.

98

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Appendices

129

Table A-1: DNA Methylation in Known Parent-of-Origin-Specific DMRs (Part 1 of 11)

Findings of journal articles that assessed cytosine methylation within known parent-of-origin-specific DMRs.

Approximately 50% allele-specific DNA methylation indicates one of the two alleles is methylated and the other allele is

unmethylated. The gene(s) associated with each analyzed region is indicated in brackets.

Sample(s) Genomic location of analyzed region (Chromosome:Mapinfo NCBI Build 36)

DNA methylation Author(s) of journal article(s) and publication year(s)

Androgenetic complete hydatidiform mole (AnCHM)

(PLAGL1)

chr6:144370175-144370176;

(PLAGL1)

chr6:144370822-144370831;

(PLAGL1)

chr6:144370994-144370995;

(PLAGL1)

chr6:144371007-144371013;

(KCNQ1OT1)

chr11:2677698-2678017;

(SNRPN)

chr15:22751097-22751347;

(PEG3)

chr19:62041857-62041864;

(PEG3)

chr19: 62043569-62043955;

(GNAS)

chr20:56862420-56862758;

(GNAS)

chr20:56898168-56898323

~0% Arima et al. (2000)162; Kou et al. (2008)163; Judson et al. (2002)164; Van den Veyver et al. (2001)165

Androgenetic complete hydatidiform mole (AnCHM)

(H19)

chr11:1977679-1978931;

(GNAS)

chr20:56848531-56848821

~100% El-Maarri et al. (2003)166; Kou et al. (2008)163; Jinno et al. (1996)167; Judson et al. (2002)164

130




Sperm (PLAGL1)

chr6:144370175-144370176;

(PLAGL1)

chr6:144370822-144370831;

(PLAGL1)

chr6:144370994-144370995;

(PLAGL1)

chr6:144371007-144371013;

(MEST)

chr7:129919324-129919502;

(KCNQ1OT1)

chr11:2677095-2677190;

(SNRPN)

chr15:22751238-22751243;

(SNRPN)

chr15:22752107-22752114;

(PEG3)

chr19:62043777-62043837

~0% Arima et al. (2000)162; Kerjean et al. (2000)168; Geuns et al. (2007)169; Glenn et al. (1996)170; El Hajj et al. (2011)171

Sperm (H19)

chr11:1977470-1978931;

(H19)

chr11:1980073-1980074;

(DLK1/MEG3)

chr14:100346971-100347214

~100% Frevel et al. (1999)172; Jinno et al. (1996)167; Chen et al. (2010)173; Hamatani et al. (2001)174; Kerjean et al. (2000)168; Geuns et al. (2007)175

131




Mature cystic ovarian teratoma (MCT) (H19)

chr11:1975666-1976289

~25% Miura et al. (1999)176

Mature cystic ovarian teratoma (MCT) (SNRPN)

chr15:22752107-22752114

~75% Miura et al. (1999)176

Oocytes (H19)

chr11:1977679-1977938;

(DLK1/MEG3)

chr14:100346971-100347214

~0% Sato et al. (2007)177; Borghol et al. (2006)178; Chen et al. (2010)173; Geuns et al. (2007)175

Oocytes (PLAGL1)

chr6:144370924-144371025;

(PLAGL1)

chr6:144371287-144371496;

(MEST)

chr7:129919324-129919502;

(KCNQ1OT1)

chr11:2677095-2677190;

(KCNQ1OT1)

chr11:2678186-2678318

~100% Sato et al. (2007)177; Arima et al. (2006)179; Geuns et al. (2007)169

132




Peripheral blood leukocytes (PBLs) with maternal uniparental disomy for chromosome 7 (mUPD7)

(GRB10)

chr7:50817553-50818298;

(MEST)

chr7:129918720-129919874

~100% Arnaud et al. (2003)21; Riesewijk et al. (1997)24

Peripheral blood leukocytes (PBLs) with paternal uniparental disomy for chromosome 7 (pUPD7)

(GRB10)

chr7:50817553-50818298;

(MEST)

chr7:129918720-129919874

~0% Arnaud et al. (2003)21; Riesewijk et al. (1997)24

Lymphoblastoid cell line (LCL) with maternal uniparental disomy for chromosome 7 (mUPD7)

(PEG10)

chr7:94124144-94124463

~100% Grabowski et al. (2003)180

Lymphoblastoid cell line (LCL) with paternal uniparental disomy for chromosome 7 (pUPD7)

(PEG10)

chr7:94124144-94124463

~0% Grabowski et al. (2003)180

Peripheral blood leukocytes (PBLs) with paternal uniparental disomy for chromosome 14 (pUPD14)

(DLK1/MEG3)

chr14:100345458-100345644;

(DLK1/MEG3)

chr14:100346971-100347328;

(MEG3)

chr14:100361297-100361356;

(MEG3)

chr14:100361746-100361908;

(MEG3)

chr14:100361948-100362060

~100% Kagami et al. (2008)181; Kagami et al. (2010)16; Rosa et al. (2005)182

133




Peripheral blood leukocytes (PBLs) with maternal uniparental disomy for chromosome 14 (mUPD14)

(DLK1/MEG3)

chr14:100345458-100345644;

(DLK1/MEG3)

chr14:100346971-100347328;

(MEG3)

chr14:100361297-100361356;

(MEG3)

chr14:100361746-100361908;

(MEG3)

chr14:100361948-100362060

~0% Kagami et al. (2008)181; Kagami et al. (2010)16; Rosa et al. (2005)182

Lymphoblastoid cell line (LCL) with maternal uniparental disomy for chromosome 15 (mUPD15); Peripheral blood leukocytes (PBLs) with maternal uniparental disomy for chromosome 15 (mUPD15); Skin fibroblasts with maternal uniparental disomy for chromosome 15 (mUPD15)

(SNRPN)

chr15:22751238-22751243;

(SNRPN)

chr15:22752107-22752114

~100% Glenn et al. (1996)170; Sutcliffe et al. (1994)183

Lymphoblastoid cell line (LCL) with paternal uniparental disomy for chromosome 15 (pUPD15); Peripheral blood leukocytes (PBLs) with paternal uniparental disomy for chromosome 15 (pUPD15); Skin fibroblasts with paternal uniparental disomy for chromosome 15 (pUPD15)

(SNRPN)

chr15:22751238-22751243;

(SNRPN)

chr15:22752107-22752114

~0% Glenn et al. (1996)170; Sutcliffe et al. (1994)183

134




A9-Maternal chromosome 1 hybrid cells (maternal chromosome 1 from a donor normal breast epithelial cell)

(DIRAS3)

chr1:68285451-68285454;

(DIRAS3)

chr1:68288917-68288920;

(DIRAS3)

chr1:68289869-68289870

~100% Yuan et al. (2003)18

A9-Paternal chromosome 1 hybrid cells (paternal chromosome 1 from a donor normal breast epithelial cell)

(DIRAS3)

chr1:68285451-68285454;

(DIRAS3)

chr1:68288917-68288920;

(DIRAS3)

chr1:68289869-68289870

~0% Yuan et al. (2003)18

A9-Maternal chromosome 6 hybrid cells (maternal chromosome 6 from a donor fibroblast)

(PLAGL1)

chr6:144370822-144370831

~100% Inoue et al. (2001)184

A9-Paternal chromosome 6 hybrid cells (paternal chromosome 6 from a donor fibroblast)

(PLAGL1)

chr6:144370822-144370831

~0% Inoue et al. (2001)184

135




Peripheral blood leukocytes (PBLs) with maternal deletion of analyzed region

(H19)

chr11:1978491-1978641;

(H19)

chr11:1979961-1980074;

(H19)

chr11:1980325-1980584

~100% Sparago et al. (2004)185; Sparago et al. (2007)186

Peripheral blood leukocytes (PBLs) with paternal deletion of analyzed region

(H19)

chr11:1978491-1978641;

(H19)

chr11:1979961-1980074;

(H19)

chr11:1980325-1980584

~0% Sparago et al. (2004)185; Sparago et al. (2007)186

Peripheral blood leukocytes (PBLs) with paternal deletion of analyzed region

(SNRPN)

chr15:22751170-22751173;

(SNRPN)

chr15:22751238-22751243;

(SNRPN)

chr15:22751544-22751547;

(SNRPN)

chr15:22752107-22752114

~100% Glenn et al. (1996)170; Beuten et al. (1996)187

Peripheral blood leukocytes (PBLs) with maternal deletion of analyzed region

(SNRPN)

chr15:22751170-22751173;

(SNRPN)

chr15:22751238-22751243;

(SNRPN)

chr15:22751544-22751547;

(SNRPN)

chr15:22752107-22752114

~0% Glenn et al. (1996)170; Beuten et al. (1996)187

136




Skin fibroblasts with paternal deletion of analyzed region; Lymphoblastoid cell line (LCL) with paternal deletion of analyzed region

(SNRPN)

chr15:22751238-22751243;

(SNRPN)

chr15:22752107-22752114

~100% Glenn et al. (1996)170

Skin fibroblasts with maternal deletion of analyzed region; Lymphoblastoid cell line (LCL) with maternal deletion of analyzed region

(SNRPN)

chr15:22751238-22751243;

(SNRPN)

chr15:22752107-22752114

~0% Glenn et al. (1996)170

137




Placenta (PLAGL1)

chr6:144370822-144370831;

(PLAGL1)

chr6:144371249-144371258

~50% (maternal allele methylated, paternal allele unmethylated)

Kamiya et al. (2000)188

Peripheral blood leukocytes (PBLs) (PEG10)

chr7:94124144-94124463;

(GNAS)

chr20:56897673-56897967;

(GNAS)

chr20:56898486-56898507

~50% (maternal allele methylated, paternal allele unmethylated)

Grabowski et al. (2003)180; Liu et al. (2000)28; Bastepe et al. (2001)189

Peripheral blood leukocytes (PBLs) (H19)

chr11:1977679-1977842;

(IGF2)

chr11:2110766-2110924;

(DLK1/MEG3)

chr14:100345458-100345644;

(DLK1/MEG3)

chr14:100346971-100347328

~50% (paternal allele methylated, maternal allele unmethylated)

El-Maarri et al. (2003)166; Schneid et al. (1993)190; Kagami et al. (2008)181; Katrincsakova et al. (2009)191

138




Brain (PEG10)

chr7:94124144-94124463;

(H19)

chr11:1978150-1978239

~50% (allele-specific) Grabowski et al. (2003)180; Hamatani et al. (2001)174

Peripheral blood leukocytes (PBLs) (H19)

chr11:1976063-1976337;

(H19)

cht11:1977470-1977938;

(IGF2)

chr11:2125947-2126095;

(PEG3)

chr19:62043569-62043955;

(GNAS)

chr20:56848498-56848821

~50% (allele-specific) Yamazawa et al. (2008)192; Vu et al. (2000)193; Kawakami et al. (2006)194; Sandovici et al. (2003)195; El-Maarri et al. (2007)196; Murrell et al. (2008)197; El-Maarri et al. (2005)198

Placenta (H19)

chr11:1976063-1976203

~50% (allele-specific) Yamazawa et al. (2008)192

Fetal brain (H19)

chr11:1976124-1976289;

(H19)

chr11:1977607-1977751

~50% (allele-specific) Vu et al. (2000)193

Embryonic ureteral tissue (H19)

chr11:1977470-1977888

~50% (allele-specific) Takai et al. (2001)199

Bladder (H19)

chr11:1977470-1977888

~50% (allele-specific) Takai et al. (2001)199; Wong et al. (2006)200

Kidney (H19)

chr11:1977470-1977888;

(H19)

chr11:1978150-1978239

~50% (allele-specific) Frevel et al. (1999)172; Hamatani et al. (2001)174

139




Colorectal tumours with retention of imprinting (ROI) of IGF2

(H19)

chr11:1977551-1977842

~50% (allele-specific) Nakagawa et al. (2001)201

Osteosarcoma tumours with retention of imprinting of IGF2

(H19)

chr11:1977587-1977938

~50% (allele-specific) Ulaner et al. (2003)202

Fetal kidney (H19)

chr11:1977607-1977751;

(H19)

chr11:1977778-1977842

~50% (allele-specific) Vu et al. (2000)193; Cui et al. (2001)203

Liver (H19)

chr11:1977626-1977888

~50% (allele-specific) Wu et al. (2008)204

Lymphoblastoid cell line (LCL) (KCNQ1OT1)

chr11:2677843-2678153

~50% (allele-specific) Beatty et al. (2006)205

140

Table A-2: Description of Blood Samples

Each row contains the sample ID, the age (in year(s) after birth (rounded down to nearest

integer)) and sex of the individual the blood sample was collected from.

Sample ID Sex (male/female) Age (year(s))

WBC1 Male 26

WBC2 Male 35

WBC3 Male 12

WBC4 Male 3

WBC5 Male 19

WBC6 Male 7

WBC7 Male 13

WBC8 Male 4

WBC9 Female 33

WBC10 Female 28

WBC11 Female 2

WBC12 Female 21

WBC13 Female 8

WBC14 Female 13

WBC15 Female 4

WBC16 Female 12

WBC17 Female 1

WBC18 Female 16

WBC19 Female 10

WBC20 Female 3

141

Table A-3: Description of Placenta Samples

Each row contains the sample ID, the age (approximate number of weeks gestation) and sex of

the conceptus the placenta sample was collected from.

Sample ID Sex (male/female) Age (~weeks gestation)

Placenta1 Male 37

Placenta2 Male 39

Placenta3 Female 39

Placenta4 Female 26

Placenta5 Female 33

Placenta6 Female 25

Placenta7 Male 38

Placenta8 Female 39

Placenta9 Male 38

Placenta10 Male 38

Placenta11 Male 27

142

Table A-4: Number of CpG Methylated Proportions Replaced with “N/A”

Sample ID Number of CpG Methylated Proportions Replaced with “N/A”

AnCHM1 9

AnCHM2 431

AnCHM3 0

WBC1 1

WBC2 77

WBC3 3

WBC4 0

WBC5 4

WBC6 2

WBC7 96

WBC8 188

WBC9 29

WBC10 23

WBC11 1

WBC12 2

WBC13 3

WBC14 1

WBC15 879

WBC16 31

MCT 0

Placenta1 44

Placenta2 3

Placenta3 141

Placenta4 1

Placenta5 96

pUPD4 13

143

Table A-5: Array CpG Sites Located in DMRs Associated with Known Imprinted Genes

List of Illumina Infinium Human Methylation27 promoter microarray CpG sites located in known parent-of-orgin-specific

DMRs (associated with known imprinted genes). The highlighted (shaded in light blue) CpG sites are paternally

methylated (as they are located in the known paternally methylated GNAS NESP55 DMR). All the other CpG sites are

maternally methylated (as they are located in known maternally methylated DMRs (associated with known imprinted

genes)).

Chromosome Mapinfo Gene associated Illumina Chromosome Mapinfo Gene associated Illumina

NCBI build 36 with CpG site TargetID NCBI build 36 with CpG site TargetID

1 68285365 DIRAS3 cg22901840 7 129918494 MEST cg13917504

1 68285433 DIRAS3 cg13697378 7 129919741 MEST cg18183281

1 68285559 DIRAS3 cg09118625 11 2676805 KCNQ1 cg27119222

1 68285651 DIRAS3 cg21808053 15 22751346 SNURF cg18506672

1 68288376 DIRAS3 cg02317907 19 62041521 ZIM2 cg17663463

1 68288565 DIRAS3 cg19114595 19 62041627 ZIM2 cg19771589

1 68288681 DIRAS3 cg11465163 19 62041816 ZIM2 cg02793099

1 68288860 DIRAS3 cg22500004 19 62041908 ZIM2 cg01656470

1 68289041 DIRAS3 cg05392265 19 62042104 ZIM2 cg27519373

1 68289053 DIRAS3 cg16148270 19 62042315 ZIM2 cg07310951

6 144370610 PLAGL1 cg25350411 19 62043025 PEG3 cg20897667

6 144370745 PLAGL1 cg07077459 19 62043252 ZIM2 cg22354595

6 144370865 PLAGL1 cg22378065 19 62043454 PEG3 cg19335327

6 144371166 PLAGL1 cg00702231 19 62043603 PEG3 cg14849423

6 144371178 PLAGL1 cg12757684 20 56848355 GNAS cg14597908

6 144371473 PLAGL1 cg08263357 20 56848772 GNAS cg05558390

6 144371522 PLAGL1 cg17895149 20 56862672 GNAS cg21988465

6 144371602 PLAGL1 cg14161241 20 56863253 GNAS cg07284407

7 50818058 GRB10 cg12903171 20 56863708 GNAS cg21625881

7 94123578 PEG10 cg19107595 20 56864058 GNAS cg14203179

7 94123896 PEG10 cg08291000 20 56896922 GNAS cg10011623

7 94124144 PEG10 cg16492735 20 56898137 GNAS cg27027803

7 94124462 PEG10 cg06695761

144

Table A-6: Candidate Maternally Methylated CpG Sites in Blood (Part 1 of 3)



1 884817 NOC2L cg19923810 3 51870732 IQCF2 cg14940420 1 19841472 NBL1 cg19136075 3 79722937 ROBO1 cg20145360

1 20338719 PLA2G2F cg11933267 3 115495602 VSIG9 cg20832020

1 20771708 CDA cg00750606 3 134601630 BFSP2 cg25249068

1 22201054 ELA3A cg10779183 3 150064527 CPA3 cg13424229

1 22836201 C1QA cg00108454 3 152174600 USH3A cg24365013

1 22842926 C1QC cg00136477 3 160473715 SCHIP1 cg24988345

1 22857181 C1QB cg22477971 3 174341611 SPATA16 cg06577725

1 22858524 C1QB cg03941108 3 185231921 HTR3D cg14483391

1 32417280 TXLNA cg21093060 3 195571421 LRRC15 cg26838900

1 41260024 FLJ23878 cg22005565 3 195572481 LRRC15 cg02121427

1 47306563 CYP4Z1 cg10751811 4 47650768 CNGA1 cg19000186

1 62558100 ANKRD38 cg00625425 4 71372315 MUC7 cg10189763

1 68285238 DIRAS3 cg24871743 4 71751036 IGJ cg25623640

1 103999392 AMY1A cg26507477 4 72423968 SLC4A4 cg19850370

1 117554836 VTCN1 cg22424746 4 89837660 NAP1L5 cg12759554

1 117555488 VTCN1 cg27446185 4 89838076 NAP1L5 cg01026744

1 119759492 HSD3B2 cg04087608 4 90252944 TIGD2 cg08377000

1 149220567 ANXA9 cg07337598 4 146778997 MMAA cg25421002

1 150947467 LCE4A cg21846488 4 153821239 FLJ32028 cg12968903

1 151222167 SPRR1A cg06101324 4 155921670 MGC27016 cg02992596

1 151240581 SPRR3 cg25856811 4 156806130 GUCY1A3 cg02210887

1 155094826 INSRR cg02049180 5 9684117 TAS2R1 cg23248452

1 155936844 FCRL3 cg25259754 5 20017186 CDH18 cg27043873

1 157440780 DARC cg23507131 5 38631977 LIFR cg01796228

1 158975863 SLAMF7 cg11721194 5 135259425 IL9 cg13842648

1 159786020 FCGR3A cg04384208 5 137447943 WNT8A cg08603768

1 165224845 MAEL cg18894878 5 149549469 SLC6A7 cg09588653

1 169484179 FMO1 cg15514848 5 151764634 NMUR2 cg03914397

1 195212153 CFHR5 cg25840094 5 168996141 DOCK2 cg02251134

1 205105925 IL20 cg23282674 6 11219880 HERV-FRD cg04304130

1 226358328 C1orf35 cg14925024 6 24597866 GPLD1 cg14023451

1 228579960 PGBD5 cg19560210 6 25938901 SLC17A1 cg03835296

2 20391158 PUM2 cg08659707 6 26608461 BTN1A1 cg07011110

2 25419299 DNMT3A cg21629895 6 30767622 NRM cg16979445

2 26634735 OTOF cg27305303 6 31026632 DPCR1 cg04645843

2 27215383 MGC44505 cg22937804 6 31188508 C6orf15 cg16150435

2 33514258 RASGRP3 cg01109219 6 32299873 NOTCH4 cg05973262

2 44356244 SLC3A1 cg02192965 6 41115500 UNC5CL cg22346765

2 45693157 FLJ10379 cg02630207 6 41411008 NCR2 cg07131544

2 87828661 PLGLB2 cg14003512 6 42231007 GUCA1A cg02091100

2 88250462 FLJ10916 cg24977027 6 47760421 GPR115 cg18841952

2 96535110 LINCR cg03102516 6 49789192 CRISP2 cg04595372

2 98352807 CNGA3 cg22241124 6 52061073 PKHD1 cg18885346

2 98353403 CNGA3 cg15954792 6 53991847 C6orf142 cg13281868

2 113752640 PAX8 cg07403255 6 54280337 TINAG cg27090087

2 135311793 ACMSD cg02812142 6 71069112 COL9A1 cg21789545

2 135312097 ACMSD cg18766847 6 74161589 DDX43 cg08124399

2 175338096 CHRNA1 cg05649009 6 88814597 SPACA1 cg13334277

2 183095173 PDE1A cg26465666 6 88912435 CNR1 cg23276695

2 191009029 FLJ20160 cg15998761 6 107185341 RTN4IP1 cg07476030

2 201688749 CFLAR cg18119407 6 131998704 ENPP3 cg08678755

2 210343863 C2orf21 cg22037121 6 159198742 LOC202459 cg11456838

2 218739143 IL8RA cg13519373 6 160852699 LPAL2 cg15398520

2 219404716 PRKAG3 cg23081213 7 5289112 SLC29A4 cg12838902

2 222996881 SGPP2 cg11300809 7 12692614 ARL4 cg09259772

2 233061180 ECEL1 cg25431974 7 27187829 HOXA10 cg00518911

2 234489672 TRPM8 cg15746445 7 37926280 EPDR1 cg27641018

3 12775469 TMEM40 cg21706946 7 44005601 WBSCR19 cg22222251

3 35658819 ARPP-21 cg05615150 7 50817425 GRB10 cg08835688

3 35658823 ARPP-21 cg12417466 7 65842232 RABGEF1 cg18884741

3 38322396 SLC22A14 cg16558203 7 72485111 FZD9 cg18438300

3 44890922 TGM4 cg09111917 7 86947735 ABCB4 cg18655915

3 46257770 CCR3 cg04111761 7 94124872 SGCE cg01169624

3 49032665 C3orf60 cg07109801 7 94124889 SGCE cg03682823

145




7 94124891 SGCE cg18139769 11 76675528 GDPD4 cg26443244 7 94374627 PPP1R9A cg15544721 11 92925818 FN5 cg00325491

7 97458059 OCM cg14260458 11 100507255 PGR cg01987509

7 107087523 SLC26A4 cg14646244 11 102251844 MMP12 cg03179866

7 122422758 TAS2R16 cg12150401 11 113434374 ZBTB16 cg25101936

7 126670180 GRM8 cg02946850 11 117204229 FXYD2 cg10997248

7 126670876 GRM8 cg09868882 11 117361217 IL10RA cg01697865

7 127042719 PAX4 cg16514843 11 117588449 AMICA1 cg23818978

7 129917832 MEST cg07427065 11 119514892 TRIM29 cg11466837

7 139175142 TBXAS1 cg14116596 12 5473803 NTF3 cg04740359

7 142178834 PRSS2 cg04958389 12 6951439 PHB2 cg10818781

7 151284387 GALNTL5 cg11091262 12 7792928 CLEC4C cg22194129

7 154059965 DPP6 cg26738880 12 8866576 A2ML1 cg27653134

8 4840683 CSMD1 cg01136458 12 12115256 BCL2L14 cg24921858

8 7308589 SPAG11 cg19787037 12 13421230 C12orf36 cg10207745

8 7330861 DEFB106A cg05810550 12 14024207 GRIN2B cg04016326

8 10419575 UNQ9391 cg01072821 12 18781846 PLCZ1 cg25573386

8 28229651 PNOC cg19391527 12 29268139 MLSTD1 cg21522988

8 38152078 BAG4 cg01607495 12 47251390 LALBA cg01726767

8 49083013 UBE2V2 cg25612480 12 48630730 AQP2 cg12650635

8 68820791 CPA6 cg21750887 12 50568077 ANKRD33 cg19948393

8 104900822 RIMS2 cg05341878 12 50866695 LOC144501 cg11051139

8 110168926 TRHR cg22268164 12 51047420 KRTHB5 cg06132342

8 118215599 SLC30A8 cg23338195 12 51047683 KRTHB5 cg21518208

8 144487860 TOP1MT cg12188860 12 51174747 KRT6A cg11471401

9 28708459 LRRN6C cg04151683 12 51383436 KRT1B cg18982568

9 33393037 AQP7 cg13246269 12 51529189 K5B cg07265310

9 35947984 OR2S2 cg26922202 12 53328560 DCD cg25372195

9 36159175 CCIN cg20870362 12 56447256 CYP27B1 cg18413900

9 103238083 ALDOB cg26181372 12 63440249 GNS cg00626466

9 116125879 ORM1 cg24552358 12 63802129 WIF1 cg20098478

9 130911826 PPP2R4 cg25587233 12 78135486 SYT1 cg22333868

9 134031851 NTNG2 cg08108641 12 98565053 FAM71C cg04282622

9 134032018 NTNG2 cg09059635 12 116284623 NOS1 cg21006686

9 136950169 FCN1 cg17357062 12 119250606 PLA2G1B cg16396488

9 137106405 OLFM1 cg08268099 12 129568871 RIMBP2 cg24272907

9 138762696 LCN6 cg11873854 13 23421698 FLJ46358 cg12682367

9 138780202 UNQ2541 cg17074151 13 26231506 GPR12 cg15726245

9 139474062 NELF cg01970325 13 45173353 NURIT cg16140179

10 43421575 ZNF485 cg25692323 14 22355205 SLC7A7 cg18960218

10 51678599 ASAH2 cg16792160 14 34169846 SNX6 cg17346022

10 61166193 SLC16A9 cg24443367 14 53493899 BMP4 cg24526899

10 90609553 ANKRD22 cg00098162 14 54976760 TBPL2 cg16036738

10 92670490 ANKRD1 cg14558138 14 69993648 ADAM21 cg05997860

10 95362435 PDE6C cg19635695 14 76362378 C14orf166B cg24887211

10 101532973 ABCC2 cg17044311 14 79747441 DIO2 cg00217795

10 111956019 MXI1 cg13017345 14 87863387 KCNK10 cg10935723

10 118947249 KCNK18 cg07637239 14 94097194 SERPINA4 cg19042947

11 1201109 MUC5B cg03609102 14 95011802 C14orf49 cg16522484

11 2447807 KCNQ1 cg01734338 14 95222519 TCL1B cg13771579

11 3077525 OSBPL5 cg11219178 14 95792705 BDKRB1 cg10238171

11 5233297 HBG2 cg10920765 14 98247530 FLJ25773 cg06906435

11 5247322 HBE1 cg08970694 15 21483851 NDN cg01989224

11 7016556 NALP14 cg25203856 15 22619883 SNRPN cg19803984

11 7066650 HNRNPG-T cg22062068 15 22751499 SNURF cg02125271

11 18226917 SAA2 cg12907644 15 22752317 SNRPN cg22555495

11 18244223 SAA1 cg15484375 15 72827787 CYP1A2 cg04968473

11 30208049 FSHB cg27420123 16 1245421 TPSD1 cg01375871

11 45072395 PRDM11 cg20227165 16 3194568 OR1F1 cg07879977

11 46914334 C11orf49 cg26198807 16 19204357 UNQ5810 cg26222045

11 56914937 PRG2 cg15357945 16 20271212 UMOD cg07456201

11 59390450 TCN1 cg20018806 16 23672805 LOC63928 cg21745164

11 59858137 MS4A6E cg08935003 16 27176649 NSMCE1 cg12391783

11 60281151 MGC35295 cg25072962 16 30031794 GDPD3 cg03297731

11 68209421 GAL cg04464446 16 31446447 ERAF cg14387505

146




16 47936080 FLJ44674 cg24217877 19 56026308 KLK15 cg26149550 16 47965498 MGC33367 cg24642468 19 56050056 KLK3 cg17687962

16 54467074 CES7 cg23092086 19 56067705 KLK2 cg07947016

16 55156689 MT4 cg01015871 19 56198085 KLK8 cg19149785

16 56721315 GTL3 cg25771195 19 56229640 KLK12 cg23894058

16 83874112 MGC22001 cg13391235 19 56452928 FLJ40235 cg21930712

16 88604267 MGC3101 cg04731384 19 56728011 SIGLEC6 cg16617137

17 1611869 SERPINF1 cg24214470 19 58486606 BIRC8 cg24454579

17 4972090 USP6 cg12954718 19 59107977 CACNG7 cg13672791

17 7062605 ACADVL cg21636577 19 59297454 OSCAR cg21098323

17 29669861 CCL8 cg27000831 19 59477160 LILRB2 cg05248470

17 36916506 KRT13 cg10742225 19 59739533 FLJ00060 cg03602500

17 38532148 BRCA1 cg06973652 19 62043946 ZIM2 cg02162069

17 39701014 SLC4A1 cg03580247 19 62044081 PEG3 cg19098268

17 42621516 CDC27 cg10910775 19 62044469 PEG3 cg18668753

17 59203720 CCDC47 cg20099806 20 1487641 SIRPD cg17423978

17 59342543 CSHL1 cg12911791 20 2465613 TMC2 cg19290962

17 59350761 GH1 cg10207917 20 2972204 GNRH2 cg07549715

17 70131151 CD300E cg04995095 20 3592151 GFRA4 cg06919203

17 71586687 LGICZ1 cg26545162 20 3660191 HSPA12B cg09458237

18 3402088 TGIF cg20322862 20 29491867 DEFB123 cg26063872

18 22474735 KCTD1 cg10539808 20 31105781 C20orf185 cg26111757

18 26997072 DSC1 cg04180953 20 31268562 C20orf71 cg01671881

18 42811817 TCEB3C cg08008233 20 41575865 L3MBTL cg20091959

18 75850362 TXNL4A cg02955504 20 41575908 L3MBTL cg23626798

19 243070 PPAP2C cg03237153 20 41576494 L3MBTL cg01071811

19 1193323 ATP5D cg07294734 20 41576510 L3MBTL cg02611863

19 3174915 BRUNOL5 cg06734812 20 41789039 FAM112A cg11398517

19 5640943 RPL36 cg17006282 20 43237051 PI3 cg02442161

19 9985267 RDH8 cg25661884 20 43609371 SPINLW1 cg10379687

19 14002098 IL27RA cg19282782 20 43609443 SPINLW1 cg23765993

19 14446209 PTGER1 cg10468702 20 44313922 CDH22 cg04640913

19 15023623 CASP14 cg01999333 20 54257492 MC3R cg19226099

19 15451069 PGLYRP2 cg17915429 20 55720103 TMEPAI cg00138126

19 15699895 OR10H2 cg24926780 20 56861337 GNAS cg00943909

19 15713574 OR10H3 cg25843439 20 56861427 GNAS cg24346429

19 16082719 RAB8A cg03621001 20 56864311 GNAS cg25283297

19 19188990 CSPG3 cg06952310 20 56864597 GNAS cg09437522

19 19234998 HAPLN4 cg21497439 20 60811330 NTSR1 cg14871138

19 38052872 SLC7A9 cg05467458 21 10012761 TPTE cg08538752

19 38555716 CEBPG cg15046693 21 10013225 TPTE cg02148834

19 40465314 HAMP cg17907567 21 18698487 PRSS7 cg20839025

19 40475132 MAG cg05055150 21 30734946 KRTAP15-1 cg16812893

19 40712072 SBSN cg23680518 21 30928577 KRTAP20-2 cg00948500

19 40746359 ATP4A cg06123346 21 40160387 PCP4 cg00895324

19 44921106 CLC cg07173760 21 42316009 C21orf121 cg18963171

19 46189062 CYP2B6 cg19756068 21 42689155 TMPRSS3 cg01214847

19 48401200 PSG4 cg27257987 22 21303200 cg10073042

19 51490709 HIF3A cg07022477 22 23947422 CRYBB2 cg10490064

19 53239260 CABP5 cg18534730 22 31183147 BPIL2 cg27195224

19 54227150 CGB2 cg15981554 22 34361187 LOC284912 cg13351406

19 54232053 CGB1 cg17164520 22 37684061 APOBEC3A cg22954818

19 55912349 SHANK1 cg11801011

147

Table A-7: Candidate Maternally Methylated CpG Sites in Placenta (Part 1 of 4)



1 3659032 CCDC27 cg10051054 2 27358950 TRIM54 cg25218351 1 15675198 ELA2B cg24030609 2 33514258 RASGRP3 cg01109219

1 20489562 FLJ32784 cg00433406 2 46600883 ATP6V1E2 cg27485921

1 22201054 ELA3A cg10779183 2 49235663 FSHR cg17327492

1 25163534 RUNX3 cg14182690 2 51108810 NRXN1 cg16279786

1 32054053 SPOCD1 cg15789095 2 68951776 BMP10 cg11308639

1 32417280 TXLNA cg21093060 2 68952789 BMP10 cg12711530

1 44642675 C1orf164 cg21578207 2 70901157 CLEC4F cg21148892

1 53972666 GLIS1 cg21142398 2 70901644 CLEC4F cg18199266

1 57093273 C8A cg16648841 2 87828661 PLGLB2 cg14003512

1 57204698 C8B cg10620457 2 88208676 FABP1 cg19910382

1 57204738 C8B cg26851374 2 88996300 cg23984130

1 60311723 C1orf87 cg18870231 2 95975323 LOC400986 cg06098051

1 65124970 JAK1 cg02755455 2 98353403 CNGA3 cg15954792

1 67039389 INSL5 cg04979933 2 99162750 MRPL30 cg15612847

1 68285238 DIRAS3 cg24871743 2 102293829 IL1RL1 cg16386158

1 68289215 DIRAS3 cg12986021 2 108271244 SULT1C1 cg13968390

1 69998451 LRRC7 cg10576828 2 118491897 FLJ10996 cg22699362

1 75850818 SLC44A5 cg27336379 2 119416152 MARCO cg11009736

1 86706913 CLCA1 cg22181664 2 128120956 GPR17 cg15869022

1 110795047 PROK1 cg18434152 2 130817910 IMP4 cg09640202

1 117554836 VTCN1 cg22424746 2 138437785 HNMT cg02906939

1 119759492 HSD3B2 cg04087608 2 144768299 GTDC1 cg27003571

1 144124550 HFE2 cg06589885 2 152299238 NEB cg16753209

1 150242190 S100A10 cg10795646 2 156988657 GPD2 cg24579667

1 150653185 CRNN cg19370451 2 168383286 B3GALT1 cg19723473

1 150902698 LCE2D cg21312148 2 175338096 CHRNA1 cg05649009

1 150914401 LCE2C cg03960217 2 183095173 PDE1A cg26465666

1 150947467 LCE4A cg21846488 2 189547719 COL3A1 cg20770175

1 150948603 LCE4A cg17542385 2 190635795 GDF8 cg18862260

1 151116362 SMCP cg21948655 2 198523309 PLCL1 cg02833180

1 151209834 SPRR4 cg08763351 2 198524422 PLCL1 cg27609819

1 151222810 SPRR1A cg04505023 2 208702965 CRYGC cg05619712

1 151270946 SPRR1B cg18780284 2 210887805 MYL1 cg11059341

1 151281905 SPRR2D cg12891678 2 218739143 IL8RA cg13519373

1 151297375 SPRR2A cg26059632 2 219990525 DES cg26259363

1 151498186 LOR cg17761453 2 220016991 APEG1 cg10062065

1 151550465 PGLYRP3 cg09448880 2 233061180 ECEL1 cg25431974

1 152430678 TPM3 cg24490338 2 237987419 COL6A3 cg00573606

1 153538047 PKLR cg21985470 2 237988139 COL6A3 cg08950375

1 159474931 NR1I3 cg06277277 2 240165186 FLJ45964 cg17977362

1 159741600 FCGR2A cg24422489 2 241739293 PPP1R7 cg08157292

1 159787215 FCGR3A cg22202141 2 242400753 NEU4 cg00684178

1 159867393 FCGR3B cg04567009 2 242594120 FLJ38379 cg16173109

1 160614634 C1orf111 cg14701962 3 3127530 IL5RA cg10159529

1 161439273 RGS5 cg10604646 3 35658819 ARPP-21 cg05615150

1 169484179 FMO1 cg15514848 3 35658823 ARPP-21 cg12417466

1 169888355 MYOC cg22077553 3 38322396 SLC22A14 cg16558203

1 180685841 RGSL2 cg06025017 3 42426880 LYZL4 cg21044104

1 180723419 RGSL1 cg01939443 3 42702164 KBTBD5 cg16842214

1 184696590 PDC cg12723191 3 46257770 CCR3 cg04111761

1 190871876 RGS13 cg05023691 3 46893270 PTHR1 cg03391568

1 196776292 ATP6V1G3 cg12958813 3 51870732 IQCF2 cg14940420

1 196874171 PTPRC cg21171615 3 52839699 ITIH4 cg10929387

1 199348309 CACNA1S cg00095526 3 52839856 ITIH4 cg17890764

1 199448506 DKFZp434B1231 cg05666713 3 79722937 ROBO1 cg20145360

1 202922001 LRRN5 cg16456919 3 109958816 RETNLB cg14659547

1 205161776 FAIM3 cg06640279 3 115495602 VSIG9 cg20832020

1 205272148 C1orf116 cg01119135 3 126135480 MUC13 cg09081544

1 207925204 HSD11B1 cg04732193 3 127760710 C3orf22 cg02765820

1 209732752 C1orf36 cg00658007 3 134601630 BFSP2 cg25249068

2 20391116 PUM2 cg01888166 3 139333801 A4GNT cg17687282

2 20391158 PUM2 cg08659707 3 141879779 TRIM42 cg12242338

2 27215383 MGC44505 cg22937804 3 150064527 CPA3 cg13424229

2 27215924 MGC44505 cg00396894 3 150065842 CPA3 cg24290574

148




3 152174600 USH3A cg24365013 7 92687787 LOC253012 cg11608424 3 185254193 HTR3C cg18271969 7 94124872 SGCE cg01169624

3 191650257 UNQ846 cg02912041 7 94124889 SGCE cg03682823

4 6040940 FLJ46481 cg00958560 7 94124891 SGCE cg18139769

4 68304786 GNRHR cg27219973 7 94953290 ASB4 cg11554605

4 68678354 TMPRSS11F cg20695587 7 97458059 OCM cg14260458

4 70950381 HTN1 cg06545504 7 99411214 AZGP1 cg19465374

4 71261606 SMR3A cg11799561 7 99412319 AZGP1 cg12019109

4 71372315 MUC7 cg10189763 7 106990684 DUS4L cg19770955

4 71751036 IGJ cg25623640 7 107231134 SLC26A3 cg04996020

4 77177150 CXCL11 cg08046471 7 107231964 SLC26A3 cg22294577

4 88462842 HSD17B13 cg24999727 7 122125815 RNF133 cg22828602

4 88790677 DMP1 cg18397653 7 126670876 GRM8 cg09868882

4 89837660 NAP1L5 cg12759554 7 127043912 PAX4 cg08886154

4 89838076 NAP1L5 cg01026744 7 128142953 NYD-SP18 cg07586911

4 123073415 TRPC3 cg18474934 7 129913072 MEST cg02490034

4 145046282 GYPE cg16998872 7 129917832 MEST cg07427065

4 145281410 GYPA cg09841009 7 135063389 SLC13A4 cg02005755

4 155702533 FGB cg18876189 7 139175142 TBXAS1 cg14116596

4 156806130 GUCY1A3 cg02210887 7 141604179 TRY1 cg14153740

5 22890461 CDH12 cg15175266 7 141604926 TRY1 cg10466917

5 41248929 C6 cg11976616 7 142539426 PIP cg26628847

5 72499443 MGC13034 cg05731779 7 150044826 GIMAP1 cg25168545

5 72500081 MGC13034 cg15905124 7 151284387 GALNTL5 cg11091262

5 131424256 IL3 cg17983064 8 7788776 DEFB4 cg02658251

5 134811085 C5orf20 cg14722162 8 7789670 DEFB4 cg22478614

5 137447943 WNT8A cg08603768 8 22074025 SFTPC cg19516009

5 146442048 PPP2R2B cg17342759 8 23376361 ENTPD4 cg24430616

5 156540696 ITK cg09453312 8 30825428 TEX15 cg19418958

5 178220323 ZNF354B cg19236727 8 38128705 STAR cg09793866

5 179951718 SCGB3A1 cg14472601 8 38445731 FGFR1 cg08722122

5 180258748 BTNL8 cg24024214 8 67568296 C8orf46 cg23704362

6 3794271 FAM50B cg01570885 8 68821882 CPA6 cg19250907

6 6568577 FLJ33708 cg18201198 8 86478130 CA1 cg17191715

6 24597866 GPLD1 cg14023451 8 87151181 PSKH2 cg19587887

6 25862169 SLC17A4 cg21627181 8 87180235 ATP6V0D2 cg00319692

6 30212531 TRIM40 cg07405796 8 87180271 ATP6V0D2 cg01248426

6 30236364 TRIM10 cg17232861 8 95291196 CDH17 cg20987610

6 32299873 NOTCH4 cg05973262 8 104900822 RIMS2 cg05341878

6 33148892 HLA-DPA1 cg13906813 8 120148806 COLEC10 cg05755779

6 33878828 MLN cg08332212 8 125252939 C8orf78 cg05412531

6 39390510 KCNK17 cg02611419 8 134273023 WISP1 cg22637941

6 41115500 UNC5CL cg22346765 8 143821003 SLURP1 cg07441143

6 41276779 TREML2 cg26928682 8 144487860 TOP1MT cg12188860

6 41411008 NCR2 cg07131544 9 5330039 RLN1 cg20789691

6 42231007 GUCA1A cg02091100 9 12683326 TYRP1 cg25989745

6 46869049 MEP1A cg16019620 9 14713496 CER1 cg01446692

6 47760421 GPR115 cg18841952 9 21156581 IFNA21 cg19982860

6 52061073 PKHD1 cg18885346 9 21208462 IFNA16 cg06479216

6 52217306 IL17F cg04063348 9 21218142 IFNA17 cg01074640

6 53991847 C6orf142 cg13281868 9 21399505 IFNA8 cg15669228

6 88912435 CNR1 cg23276695 9 34700657 CCL21 cg07269146

6 109867524 SMPD2 cg20811607 9 35032395 MGC41945 cg05000446

6 124000883 TRDN cg14462830 9 36159175 CCIN cg20870362

6 131998704 ENPP3 cg08678755 9 100745613 COL15A1 cg20503329

6 134251673 TCF21 cg10771262 9 103397922 PPP3R2 cg15765694

6 135313282 ALDH8A1 cg09533063 9 113597060 C9orf84 cg06643227

6 160247992 MAS1 cg08784110 9 113597374 C9orf84 cg13314167

7 989201 CYP2W1 cg15914863 9 116125879 ORM1 cg24552358

7 20653748 ABCB5 cg22066521 9 123963104 NDUFA8 cg01536400

7 27187829 HOXA10 cg00518911 9 138237697 LHX3 cg14091657

7 37926280 EPDR1 cg27641018 10 4994250 AKR1C1 cg07639198

7 44147129 MYL7 cg23370883 10 13584654 C10orf30 cg23114594

7 44196417 GCK cg15001372 10 17211701 CUBN cg10707565

7 75257240 CCL26 cg05556717 10 61819563 ANK3 cg12354377

149




10 85991134 LRRC21 cg17827767 12 47251390 LALBA cg01726767 10 88418237 LDB3 cg08300860 12 48630730 AQP2 cg12650635

10 90609553 ANKRD22 cg00098162 12 51001958 KRTHB3 cg16791508

10 90702747 ACTA2 cg07436807 12 51066060 KRTHB4 cg18153060

10 95362185 PDE6C cg04298323 12 51299598 K6IRS3 cg07017706

10 95362435 PDE6C cg19635695 12 51457458 KRT2B cg11465372

10 98109069 TMEM10 cg06825166 12 51495029 KRT4 cg12610744

10 101532973 ABCC2 cg17044311 12 53043844 GPR84 cg21969640

10 102079671 PKD2L1 cg10365880 12 53177758 HEM1 cg17605084

10 115501494 C10orf81 cg10368842 12 53177901 HEM1 cg16509569

10 126480089 KIAA0157 cg26147480 12 53315414 LACRT cg07643942

10 127503180 UROS cg19346193 12 54911376 SLC39A5 cg03343942

10 127503243 UROS cg04117029 12 55729889 MYO1A cg09541248

11 552366 RASSF7 cg14896003 12 63802129 WIF1 cg20098478

11 831335 TSPAN4 cg03116740 12 69838603 TSPAN8 cg12965512

11 2401968 TRPM5 cg07882535 12 78135486 SYT1 cg22333868

11 2499264 KCNQ1 cg17229197 12 85756642 MGAT4C cg18344063

11 2499438 KCNQ1 cg12949760 12 103847526 SLC41A2 cg23855818

11 2550433 KCNQ1 cg08007665 13 23421698 FLJ46358 cg12682367

11 2550536 KCNQ1 cg26750319 13 30207799 ALOX5AP cg08529529

11 2550729 KCNQ1 cg20751395 13 31787023 BRCA2 cg12836863

11 3077862 OSBPL5 cg12514506 13 42043414 TNFSF11 cg24222324

11 3623059 ART1 cg21407055 13 42252674 FLJ40919 cg07409200

11 5205142 HBB cg14544583 13 42494577 DNAJC15 cg11679069

11 6419028 HPX cg11547724 13 44460280 KIAA1704 cg22539738

11 11988084 DKK3 cg25787984 13 69581128 KLHL1 cg20349377

11 18214899 SAA4 cg22587758 13 102516931 SLC10A2 cg18312429

11 19181235 CSRP3 cg14704941 13 107719053 TNFSF13B cg09646392

11 22646390 GAS2 cg06493930 13 113369467 GRK1 cg09034896

11 36544793 RAG1 cg11494699 14 20306951 FAM12B cg15842430

11 36546820 RAG1 cg10399228 14 20537342 SLC39A2 cg05654163

11 60139937 MGC39681 cg07484450 14 22911594 IL17E cg14366598

11 60858741 DAK cg25406518 14 23879599 RIPK3 cg10318258

11 61278578 C11orf9 cg04237003 14 53493899 BMP4 cg24526899

11 61279074 C11orf9 cg22627427 14 59781450 PPM1A cg17884373

11 61794235 SCGB2A2 cg22862656 14 73885069 C14orf115 cg21207436

11 62539699 SLC22A8 cg06917325 14 76362378 C14orf166B cg24887211

11 64079466 SLC22A11 cg09705062 14 76362486 C14orf166B cg08675585

11 66245214 SPTBN2 cg04985144 14 77245223 ALKBH cg20385229

11 67047602 CABP2 cg18138484 14 93927028 SERPINA1 cg24621042

11 75105846 MOGAT2 cg10585962 14 94147310 SERPINA3 cg16507522

11 76675528 GDPD4 cg26443244 15 21361373 MKRN3 cg16131766

11 88550066 TYR cg03417466 15 21362298 MKRN3 cg23234999

11 92925818 FN5 cg00325491 15 22751499 SNURF cg02125271

11 102251844 MMP12 cg03179866 15 22752317 SNRPN cg22555495

11 117205061 FXYD2 cg25894551 15 23073264 HBII-438B cg18499731

11 117361080 IL10RA cg26661481 15 28474184 CHRFAM7A cg06319346

11 117361217 IL10RA cg01697865 15 40077231 PLA2G4E cg15228639

11 117720322 CD3G cg15880738 15 41300855 EPB42 cg18431127

11 118524594 ABCG4 cg03222066 15 46200510 SLC24A5 cg01497576

11 118559919 PDZD3 cg05461276 15 46285777 SLC12A1 cg20226593

11 122214681 CRTAM cg10977115 15 69807614 THSD4 cg04616566

11 123986824 PANX3 cg08191915 15 72446954 CYP11A1 cg06285340

11 125154444 C11orf38 cg23743472 15 72827787 CYP1A2 cg04968473

11 131038494 C11orf39 cg25943276 15 72828439 CYP1A2 cg09207718

12 4569879 DYRK4 cg09418321 15 98901965 LASS3 cg00729708

12 5473803 NTF3 cg04740359 16 1851526 MGC35212 cg00892393

12 6423750 TNFRSF7 cg06495803 16 2741794 SRRM2 cg06736444

12 6819521 GNB3 cg05484458 16 10697202 FLJ32871 cg18609562

12 9252121 PZP cg01714932 16 20323467 PDILT cg04491443

12 11354646 PRB4 cg14076161 16 31312602 ITGAD cg02164442

12 14024207 GRIN2B cg04016326 16 47965498 MGC33367 cg24642468

12 14740535 GUCY2C cg18754342 16 55156689 MT4 cg01015871

12 18781846 PLCZ1 cg25573386 16 70645745 HP cg06172871

12 29268139 MLSTD1 cg21522988 16 70654445 HPR cg09584711

150




16 83874112 MGC22001 cg13391235 19 61202777 NALP5 cg26264314 17 7062572 ACADVL cg24825722 19 62043946 ZIM2 cg02162069

17 7062605 ACADVL cg21636577 19 62044081 PEG3 cg19098268

17 7931430 ALOX12B cg03742272 19 62044469 PEG3 cg18668753

17 16816676 TNFRSF13B cg23274244 20 155338 DEFB129 cg00769470

17 23823416 SLC13A2 cg11653858 20 186156 DEFB32 cg18239253

17 28341649 SPACA3 cg26829529 20 3660191 HSPA12B cg09458237

17 29636788 CCL11 cg24870391 20 23381349 CST11 cg06489008

17 29707167 CCL13 cg02706575 20 23496991 CST9L cg15210427

17 31332250 CCL16 cg10190509 20 23755958 CST2 cg01237132

17 31337750 CCL14 cg09256683 20 29492554 DEFB123 cg19241311

17 31354012 CCL15 cg26548883 20 30061355 C20orf160 cg11801374

17 31616166 TBC1D3C cg02601403 20 31105781 C20orf185 cg26111757

17 35975201 CCR7 cg17067993 20 31268562 C20orf71 cg01671881

17 36113460 KRT24 cg24340657 20 31335013 C20orf114 cg22789545

17 36113623 KRT24 cg23737768 20 36407859 LBP cg18979491

17 36165064 KRT25A cg22983092 20 41575865 L3MBTL cg20091959

17 36210026 KRT25D cg20484002 20 41575908 L3MBTL cg23626798

17 36997052 KRT14 cg01602596 20 41576494 L3MBTL cg01071811

17 38306255 G6PC cg26673195 20 41576510 L3MBTL cg02611863

17 43389454 ATAD4 cg10878307 20 41620770 SGK2 cg21685427

17 44226904 TTLL6 cg08137716 20 42777411 WISP2 cg03562120

17 53669778 LPO cg12032049 20 43766147 WFDC10B cg00690280

17 57810848 EFCAB3 cg07292816 20 43950634 C20orf165 cg21023770

17 64462140 ABCA8 cg21660392 20 56861225 GNAS cg17414107

17 69788532 DNAI2 cg11856697 20 56861337 GNAS cg00943909

17 69875229 GPR142 cg03803009 20 56861427 GNAS cg24346429

17 71975276 AANAT cg09382492 20 56864311 GNAS cg25283297

18 22474735 KCTD1 cg10539808 20 56898834 GNAS cg25983380

18 22699132 C18orf16 cg00729275 20 58063710 FLJ33860 cg09076077

18 22699316 C18orf16 cg15976539 20 62208641 NPBWR2 cg21628553

18 28024079 MEP1B cg01941619 20 62266251 MYT1 cg16772207

18 42816134 TCEB3B cg20879768 21 29314090 C21orf6 cg05406101

18 59734679 SERPINB10 cg23696618 21 30734946 KRTAP15-1 cg16812893

19 4132854 SIRT6 cg09936839 21 30797319 KRTAP19-5 cg07374637

19 8670345 MGC33407 cg18931750 22 18091327 GP1BB cg07359545

19 14002098 IL27RA cg19282782 22 20710839 cg00013618

19 14490905 DNAJB1 cg15712267 22 21303200 cg10073042

19 15451069 PGLYRP2 cg17915429 22 22907315 SUSD2 cg03599338

19 18890194 DDX49 cg14757492 22 25344415 CRYBB1 cg00757952

19 41191679 FLJ36445 cg21550442 22 29290857 GAL3ST1 cg09022808

19 44921106 CLC cg07173760 22 30981345 SLC5A4 cg21578906

19 45611085 PRX cg26200585 22 31082769 RFPL3 cg13005002

19 46884924 CEACAM7 cg07297178 22 31183844 BPIL2 cg14789590

19 47073790 CD79A cg04790874 22 34442848 APOL5 cg03128832

19 47623519 LIPE cg14679230 22 35734834 MGC35206 cg22088368

19 53500080 FLJ32926 cg11521325 22 36548891 GALR3 cg22975712

19 54091600 NUCB1 cg21252483 22 36782815 PRKCABP cg23621115

19 55123237 ATF5 cg23878206 22 38619484 FLJ25421 cg08245789

19 56728011 SIGLEC6 cg16617137 22 40856563 CYP2D6 cg10840135

19 56946034 FPR1 cg15811427

151

Table A-8: Candidate Paternally Methylated CpG Sites in Blood



1 15784289 AGMAT cg17385448 11 92342296 MTNR1B cg15842276 1 35811746 TFAP2E cg26372517 12 7234287 PEX5 cg15754084

1 111963591 RAP1A cg23301687 13 30671806 B3GTL cg21418052

1 153557921 C1orf104 cg22234962 13 94051677 ITR cg09582042

1 221633332 C1orf65 cg27202708 14 38804962 CTAGE5 cg13277939

1 221633417 C1orf65 cg05333568 14 80491864 TSHR cg09721659

2 27659126 ZNF512 cg01464985 15 32875399 ACTC cg06048973

2 101974624 IL1R2 cg17142183 15 51838835 WDR72 cg18613421

2 172572583 MAP1D cg16449464 15 72969183 MPI cg13828047

3 47492823 SCAP cg26614073 15 88344817 ZNF710 cg01185080

3 126414646 SLC12A8 cg14391622 16 704795 METRN cg11027330

4 110844529 CASP6 cg17714799 16 3433998 ZNF597 cg14654875

4 178601336 AGA cg00398048 16 8965866 USP7 cg12914014

5 134763443 H2AFY cg01550148 16 10943234 DEXI cg27425675

5 137637734 GFRA3 cg20178764 16 64957821 CDH5 cg08872742

5 139992912 CD14 cg25358289 16 65517230 RRAD cg19428417

5 178353920 GRM6 cg15674997 16 68890379 DDX19B cg14244577

5 179153151 LTC4S cg16361890 16 79829782 BCMO1 cg22947000

6 26472772 BTN3A2 cg14345882 16 79830001 BCMO1 cg17465631

6 153494647 RGS17 cg23651356 17 7225386 TNK1 cg25499099

6 158163595 SNX9 cg20179697 17 7883131 ALOX15B cg15799267

7 5051647 RBAK cg06914598 17 24531793 MYO18A cg00426498

7 126820931 LOC168850 cg08137040 17 25729371 CPD cg07156669

7 127668516 LEP cg19594666 17 45901117 CHAD cg06958829

9 2828508 KIAA0020 cg24471894 17 75428659 CBX4 cg04398978

9 5294611 RLN2 cg02875297 17 76884479 MGC15523 cg00466249

10 99249711 UBTD1 cg17296078 17 76884481 MGC15523 cg06850526

10 125415252 GPR26 cg07036530 18 54681282 ZNF532 cg12406559

10 125641024 CPXM2 cg09619146 19 1463314 ADAMTSL5 cg04601137

10 135011037 PRAP1 cg10742801 19 40939917 HSPB6 cg15125472

11 1976144 H19 cg17769238 19 41215245 CLIPR-59 cg06432655

11 1977136 H19 cg02657360 19 46417172 AXL cg14892768

11 2111008 IGF2 cg22956483 19 54707592 FCGRT cg15528736

11 2847033 KCNQ1DN cg13081704 20 56847934 GNAS cg25268451

11 5667230 TRIM22 cg12461141 20 56849616 GNAS cg24975842

11 32408763 WT1 cg01693350 20 56849901 GNAS cg18619398

11 65444453 Bles03 cg13547237 22 39405619 GPR24 cg21342728

11 65444468 Bles03 cg10467098 22 49333679 KLHDC7B cg18533225

11 85763271 FLJ23514 cg16404106

152

Table A-9: Candidate Paternally Methylated CpG Sites in Placenta



1 4613868 AJAP1 cg20959866 12 47975479 PRPH cg09595479 1 21867684 RAP1GA1 cg22396755 12 52696843 HOXC4 cg21487207

1 26360515 ZNF593 cg11418559 13 30671806 B3GTL cg21418052

1 26361575 ZNF593 cg18107827 13 32489130 KL cg23282559

1 36322313 TEKT2 cg24813212 13 94051677 ITR cg09582042

1 54392033 CDCP2 cg26185508 14 23873647 ADCY4 cg16761581

1 85498480 C1orf52 cg05654164 14 23878913 RIPK3 cg20822579

1 109005848 C1orf59 cg00328227 14 80491864 TSHR cg09721659

1 153557921 C1orf104 cg22234962 14 102459567 AMN cg01155039

1 204874797 DYRK3 cg09328024 15 51838835 WDR72 cg18613421

1 221633332 C1orf65 cg27202708 15 76419647 CRABP1 cg16703647

2 6935238 RSAD2 cg18201077 15 76419725 CRABP1 cg11200929

2 72997307 EMX1 cg26898166 15 76419916 CRABP1 cg17133183

2 172572583 MAP1D cg16449464 15 76420510 CRABP1 cg19777470

3 112743473 CD96 cg04039397 15 76420717 CRABP1 cg20550118

3 126414646 SLC12A8 cg14391622 15 82974955 SCAND2 cg17866455

3 142432606 ACPL2 cg00400028 16 360113 MRPL28 cg12437481

4 1981675 WHSC2 cg14217157 16 704795 METRN cg11027330

4 3504643 LRPAP1 cg25201363 16 3433998 ZNF597 cg14654875

4 39875171 RHOH cg11903057 16 8965866 USP7 cg12914014

4 108861860 PAPSS1 cg09191232 16 45474997 GPT2 cg18587271

5 151118744 ATOX1 cg06746171 16 66007362 ZDHHC1 cg11657615

6 35217099 TCP11 cg22407458 16 68155671 NFAT5 cg11147886

6 158163595 SNX9 cg20179697 16 73707957 LDHD cg03991512

7 37922801 SFRP4 cg08261094 16 83239008 C16orf44 cg23617760

7 43764312 BLVRA cg17571291 17 19822224 AKAP10 cg11630242

7 127668168 LEP cg12782180 17 24968711 CORO6 cg06038133

7 127668516 LEP cg19594666 17 25729371 CPD cg07156669

7 139409708 PARP12 cg07937272 17 38166489 RAMP2 cg14436761

7 150848513 RHEB cg03998173 17 38250104 AOC2 cg19317715

7 150848816 RHEB cg21134096 17 39574438 C17orf53 cg25425074

8 41287005 SFRP1 cg06166767 17 45858056 FLJ20920 cg14672994

9 33438382 AQP3 cg26624914 17 71648573 FOXJ1 cg01294702

9 129587031 CDK9 cg08999352 17 72046145 CYGB cg21301440

9 131468359 PRRX2 cg04713521 17 76884479 MGC15523 cg00466249

10 44201060 CXCL12 cg18618334 17 76884481 MGC15523 cg06850526

10 44815977 ZNF22 cg00899659 17 78267247 FN3KRP cg14688272

10 64245804 EGR2 cg19355190 18 54681282 ZNF532 cg12406559

10 73393227 CHST3 cg04268405 19 1463314 ADAMTSL5 cg04601137

10 90333188 C10orf59 cg06981182 19 3087710 GNA15 cg26482939

10 95351376 RBP4 cg12936747 19 4230441 SHD cg26646370

10 135043202 PAOX cg18361093 19 15204174 ABHD9 cg05488632

11 1976144 H19 cg17769238 19 17766332 B3GNT3 cg23771661

11 1977136 H19 cg02657360 19 41215245 CLIPR-59 cg06432655

11 2110567 IGF2 cg02807948 19 45016586 DYRK1B cg10294836

11 2111008 IGF2 cg22956483 19 45016824 DYRK1B cg18851831

11 2122537 IGF2AS cg11005826 19 45774154 SHKBP1 cg09381003

11 2510986 KCNQ1 cg16465939 19 47329470 POU2F2 cg22054191

11 2511159 KCNQ1 cg27491887 19 50693586 FLJ40125 cg00116838

11 5667230 TRIM22 cg12461141 20 34603023 MYL9 cg21671476

11 46310677 DGKZ cg18765542 20 44469045 ELMO2 cg14261863

11 47692758 AGBL2 cg01888601 20 56847934 GNAS cg25268451

11 56860207 SSRP1 cg01511567 20 56849616 GNAS cg24975842

11 65081734 LTBP3 cg08965235 20 56849901 GNAS cg18619398

12 5023549 KCNA5 cg20792062 20 60743500 SLCO4A1 cg09210315

12 7739360 GDF3 cg15992730 21 43973826 C21orf124 cg21755709

12 7916762 SLC2A14 cg05521696 21 44056660 LOC284837 cg00546897

12 47658741 WNT1 cg21948783 22 19699610 P2RXL1 cg19592945

153

Table A-10: PCR Conditions for Bisulfite Pyrosequencing

Reagent (first column)/DMR

(first row)

DIRAS3 KvDMR1 H19DMR IG-

DMR

NAP1L5 ZNF597 AXL RIMS2

Distilled water (μL) 17.25 19.25 18.25 17.25 18

10*BF (μL) 2.5

25 mM magnesium chloride

(μL)

2 0 1 2 1.25

10 mM dNTP solution (μL) 0.5

10 μM M13-Biotin Universal

Primer Complex (μL)

0.5

10 μM solution of forward

primer (μL)

0.5

10 μM solution of reverse

primer (μL)

0.5

HotStar Taq (μL) 0.25

Sodium bisulfite treated DNA

(μL)

1

154

Table A-11: PCR Cycling Conditions for Bisulfite Pyrosequencing

Note: stages follow each other in succession. N/A stands for not applicable.

Stage (first

column)/DMR

(first row)

DIRAS3 KvDMR1 H19DMR IG-DMR NAP1L5 ZNF597 AXL RIMS2

1 95°C for 15 minutes

2 30 cycles

of (95°C

for 30

seconds,

65°C

(decrease

temp after

first cycle

by 0.5°C

every 1

cycle) for

30 seconds,

72°C for

30

seconds)

10 cycles of

(95°C for

30 seconds,

57°C

(decrease

temp after

first cycle

by 0.5°C

every 1

cycle) for

30 seconds,

72°C for 30

seconds)

10 cycles of

(95°C for

30 seconds,

60°C

(decrease

temp after

cycle 1 by

0.5°C every

1 cycle) for

30 seconds,

72°C for 30

seconds)

10 cycles

of (95°C

for 20

seconds,

55°C

(decrease

temp after

cycle 1 by

0.5°C

every 1

cycle) for

20 seconds,

72°C for

20

seconds)

50 cycles

of (95°C

for 30

seconds,

65°C for

30

seconds,

72°C for

45

seconds)

10 cycles

of (95°C

for 30

seconds,

60°C

(decrease

temp after

first cycle

by 0.5°C

every 1

cycle) for

30 seconds,

72°C for

30

seconds)

50 cycles

of (95°C

for 30

seconds,

61°C for

30

seconds,

72°C for

30

seconds)

35 cycles

of (95°C

for 30

seconds,

56°C for

30

seconds,

72°C for

30

seconds)

3 20 cycles

of (95°C

for 30

seconds,

50°C for

30 seconds,

72°C for

30

seconds)

30 cycles of

(95°C for

30 seconds,

52°C for 30

seconds,

72°C for 30

seconds)

38 cycles of

(95°C for

30 seconds,

55°C for 30

seconds,

72°C for 30

seconds)

30 cycles

of (95°C

for 20

seconds,

50°C for

20 seconds,

72°C for

20

seconds)

72°C for

10

minutes

30 cycles

of (95°C

for 30

seconds,

55°C for

30 seconds,

72°C for

30

seconds)

72°C for

10

minutes

72°C for

7 minutes

4 72°C for 7 minutes N/A 72°C for 7

minutes

N/A N/A

155

Table A-12: Primer Sequences for PCR for Bisulfite Pyrosequencing

The underlined sequence was added to the PCR primers to enable the PCR products (one strand of the PCR product

specifically) to anneal to the Biotin-M13 Universal Primer complex.

DMR Forward primer (5’-3’) Reverse primer (5’-3’)

DIRAS3 CCAGGGTTTTCCCAGTCACGACGATTTTTTTGTGGGGTTTGAA CCAACTTTAACTCCAAAAAACA

Kv GTGATGTGTTTATTATT CGCCAGGGTTTTCCCAGTCACGACCTAAACRCCCACAAACCTCCA

H19 TGAGTGTTTTATTTTTAGATGATTTT CGCCAGGGTTTTCCCAGTCACGACACAATACAAACTCACACATCACAAC

IG- GTTTTATTATTGAATTGGGTTTGTTAGTA CGCCAGGGTTTTCCCAGTCACGACTCAAAACAACTCAAATCCTTTATAAC

NAP1L5 GGTTTTGTATTAGAGTTGGTTTAGAGAGAT CGCCAGGGTTTTCCCAGTCACGACAAACACCTCCAACAACTACTAACACTCC

ZNF597 GGTTAGGTTTAGAAAGGGGTTTAT CGCCAGGGTTTTCCCAGTCACGACCCTCTCCCAACTACCCAATAACTA

AXL TTGGGAGTTTACTAATTTTTGAGGAAAG CGCCAGGGTTTTCCCAGTCACGACCATCACTCACCCCTAAAAACCATA

RIMS2 CGCCAGGGTTTTCCCAGTCACGACTTTTAGTTGGGAATGTTTAAAAAG CCCCAATCAAATTCCATATTTC

156

Table A-13: Primer Sequences For Bisulfite Pyrosequencing Reactions

DMR Sequencing primer (5’-3’) Amplicon size of PCR product to

be pyrosequenced (base pairs)

Number of CpG sites

assayed by pyrosequencing

DIRAS3 CAAAAAACAAAAACTACTAA 95 3

Kv GTGATGTGTTTATTATT 123 5

H19 GTGGTTTGGGTGATT 148 3

IG TGAATTGGGTTTGTTAGTAG 101 5

NAP1L5 TTTTGAGGATGAGGTAAGT 298 6

ZNF597 TTTGATAGGAGTTGTAGAAA 316 4

AXL GGTGTTTTAGGATGGGTAG 133 4

RIMS2 CCTAAAAAATTCAATACCC 197 2

157

Table A-14: PCR Conditions for Sodium Bisulfite Sequencing

Reagent Volume (μL)

Distilled water (μL) 19.75

10*BF (μL) 2.5


10 μM solution of forward primer (μL) 0.5

10 μM solution of reverse primer (μL) 0.5


Sodium bisulfite treated DNA (μL) 1

158

Table A-15: PCR Cycling Conditions for Sodium Bisulfite Sequencing

Note: stages follow each other in succession.

Stage Program


2 10 cycles of (95°C for 30 seconds, 60°C (decrease temp after first cycle by 0.5°C every 1 cycle) for 30 seconds, 72°C

for 30 seconds)

3 30 cycles of (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds)


159

Table A-16: Primer Sequences for PCR for Sodium Bisulfite Sequencing

Forward primer (5’-3’) Reverse primer (5’-3’)

GAGGTAGGGGTGTTGAGAAG CCCCACACCTACCATCAT

160

Table A-17: PCR Conditions for SNP (rs1051008) Pyrosequencing

Reagent Volume (μL)

Distilled water (μL) 18.25

10*BF (μL) 2.5

25 mM magnesium chloride (μL) 0.5


10 μM M13-Biotin Universal Primer Complex (μL) 0.5

10 μM solution of forward primer (μL) 0.5

10 μM solution of reverse primer (μL) 0.5


DNA (μL) 2

161

Table A-18: PCR Cycling Conditions for SNP (rs1051008) Pyrosequencing

Note: stages follow each other in succession.

Stage Program


2 40 cycles of (95°C for 20 seconds, 54°C for 20 seconds, 72°C for 20 seconds)


162

Table A-19: Primer Sequences for PCR for SNP (rs1051008) Pyrosequencing

The underlined sequence was added to the PCR primers to enable the PCR products (one strand of the PCR product

specifically) to anneal to the Biotin-M13 Universal Primer complex.

Forward primer (5’-3’) Reverse primer (5’-3’)

CCAGGGTTTTCCCAGTCACGACCTTTGCTGCATTCTGCCTCTCT GGAAAGGAGGCATCCCTAAT

163

Table A-20: Primer Sequence for SNP (rs1051008) Pyrosequencing Reactions

Sequencing primer (5’-3’) Amplicon size of PCR product to be pyrosequenced (base pairs)

GTGCCTAGAACTATAAGATT 290

164

Table A-21: Overlapping Candidate Maternally Methylated CpG sites



1 22201054 ELA3A cg10779183 7 151284387 GALNTL5 cg11091262 1 32417280 TXLNA cg21093060 8 104900822 RIMS2 cg05341878

1 68285238 DIRAS3 cg24871743 8 144487860 TOP1MT cg12188860

1 117554836 VTCN1 cg22424746 9 36159175 CCIN cg20870362

1 119759492 HSD3B2 cg04087608 9 116125879 ORM1 cg24552358

1 150947467 LCE4A cg21846488 10 90609553 ANKRD22 cg00098162

1 169484179 FMO1 cg15514848 10 95362435 PDE6C cg19635695

2 20391158 PUM2 cg08659707 10 101532973 ABCC2 cg17044311

2 27215383 MGC44505 cg22937804 11 76675528 GDPD4 cg26443244

2 33514258 RASGRP3 cg01109219 11 92925818 FN5 cg00325491

2 87828661 PLGLB2 cg14003512 11 102251844 MMP12 cg03179866

2 98353403 CNGA3 cg15954792 11 117361217 IL10RA cg01697865

2 175338096 CHRNA1 cg05649009 12 5473803 NTF3 cg04740359

2 183095173 PDE1A cg26465666 12 14024207 GRIN2B cg04016326

2 218739143 IL8RA cg13519373 12 18781846 PLCZ1 cg25573386

2 233061180 ECEL1 cg25431974 12 29268139 MLSTD1 cg21522988

3 35658819 ARPP-21 cg05615150 12 47251390 LALBA cg01726767

3 35658823 ARPP-21 cg12417466 12 48630730 AQP2 cg12650635

3 38322396 SLC22A14 cg16558203 12 63802129 WIF1 cg20098478

3 46257770 CCR3 cg04111761 12 78135486 SYT1 cg22333868

3 51870732 IQCF2 cg14940420 13 23421698 FLJ46358 cg12682367

3 79722937 ROBO1 cg20145360 14 53493899 BMP4 cg24526899

3 115495602 VSIG9 cg20832020 14 76362378 C14orf166B cg24887211

3 134601630 BFSP2 cg25249068 15 22751499 SNURF cg02125271

3 150064527 CPA3 cg13424229 15 22752317 SNRPN cg22555495

3 152174600 USH3A cg24365013 15 72827787 CYP1A2 cg04968473

4 71372315 MUC7 cg10189763 16 47965498 MGC33367 cg24642468

4 71751036 IGJ cg25623640 16 55156689 MT4 cg01015871

4 89837660 NAP1L5 cg12759554 16 83874112 MGC22001 cg13391235

4 89838076 NAP1L5 cg01026744 17 7062605 ACADVL cg21636577

4 156806130 GUCY1A3 cg02210887 18 22474735 KCTD1 cg10539808

5 137447943 WNT8A cg08603768 19 14002098 IL27RA cg19282782

6 24597866 GPLD1 cg14023451 19 15451069 PGLYRP2 cg17915429

6 32299873 NOTCH4 cg05973262 19 44921106 CLC cg07173760

6 41115500 UNC5CL cg22346765 19 56728011 SIGLEC6 cg16617137

6 41411008 NCR2 cg07131544 19 62043946 ZIM2 cg02162069

6 42231007 GUCA1A cg02091100 19 62044081 PEG3 cg19098268

6 47760421 GPR115 cg18841952 19 62044469 PEG3 cg18668753

6 52061073 PKHD1 cg18885346 20 3660191 HSPA12B cg09458237

6 53991847 C6orf142 cg13281868 20 31105781 C20orf185 cg26111757

6 88912435 CNR1 cg23276695 20 31268562 C20orf71 cg01671881

6 131998704 ENPP3 cg08678755 20 41575865 L3MBTL cg20091959

7 27187829 HOXA10 cg00518911 20 41575908 L3MBTL cg23626798

7 37926280 EPDR1 cg27641018 20 41576494 L3MBTL cg01071811

7 94124872 SGCE cg01169624 20 41576510 L3MBTL cg02611863

7 94124889 SGCE cg03682823 20 56861337 GNAS cg00943909

7 94124891 SGCE cg18139769 20 56861427 GNAS cg24346429

7 97458059 OCM cg14260458 20 56864311 GNAS cg25283297

7 126670876 GRM8 cg09868882 21 30734946 KRTAP15-1 cg16812893

7 129917832 MEST cg07427065 22 21303200 cg10073042

7 139175142 TBXAS1 cg14116596

165

Table A-22: Overlapping Candidate Paternally Methylated CpG sites



1 153557921 C1orf104 cg22234962 15 51838835 WDR72 cg18613421 1 221633332 C1orf65 cg27202708 16 704795 METRN cg11027330

2 172572583 MAP1D cg16449464 16 3433998 ZNF597 cg14654875

3 126414646 SLC12A8 cg14391622 16 8965866 USP7 cg12914014

6 158163595 SNX9 cg20179697 17 25729371 CPD cg07156669

7 127668516 LEP cg19594666 17 76884479 MGC15523 cg00466249

11 1976144 H19 cg17769238 17 76884481 MGC15523 cg06850526

11 1977136 H19 cg02657360 18 54681282 ZNF532 cg12406559

11 2111008 IGF2 cg22956483 19 1463314 ADAMTSL5 cg04601137

11 5667230 TRIM22 cg12461141 19 41215245 CLIPR-59 cg06432655

13 30671806 B3GTL cg21418052 20 56847934 GNAS cg25268451

13 94051677 ITR cg09582042 20 56849616 GNAS cg24975842

14 80491864 TSHR cg09721659 20 56849901 GNAS cg18619398

166

Table A-23: Candidate DMCpGs That May Represent Components of Known DMRs

Chromosome Mapinfo (NCBI

build 36/hg18)

Illumina TargetID Distance to closest

known parent-of-origin-

specific DMR associated

with known imprinted

genes (base pairs)

Name of closest known

parent-of-origin-specific

DMR associated with

known imprinted genes

Is the closest known parent-

of-origin-specific DMR

associated with known

imprinted genes maternally

or paternally methylated?

1 68285238 cg24871743 < 300 DIRAS3

94124872 cg01169624

94124889 cg03682823

94124891 cg18139769

< 500 PEG10 7

129917832 cg07427065 < 900 MEST

Maternally

11 1977136 cg02657360 < 600 IC1 (H19DMR)

11 2111008 cg22956483 IGF2 DMR2

Paternally

22751499 cg02125271

< 100

15

22752317 cg22555495 < 300

SNRPN Maternally

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