C h a p t e r
8
The Human Epigenome:
Implications for the
Understanding of Human
Disease
Maria Berdasco
INTRODUCTION
Epigenetic processes, defined as the heritable patterns of
gene expression that do not involve changes in the
sequence of the genome, and their effects on gene
repression are increasingly understood to be such a way
of modulating phenotype transmission and develop-
ment. The patterns of DNA methylation, histone modi-
fications, microRNAs, and several chromatin-related
proteins of sick cells usually differ from those of healthy
cells, highlighting the importance of epigenetic regula-
tion in most human pathologies. The aim of the present
review is to provide an overview of how epigenetic factors
contribute to the development of human diseases such
as abnormal imprinting-causative pathologies, cancer
malignancies, as well as autoimmune, cardiovascular,
and neurological disorders. These studies have provided
extensive information about the mechanisms that con-
tribute to the phenotype of human diseases, but also
provided opportunities for therapy.
EPIGENETIC REGULATION
OF THE GENOME
The primary goal of the Human Genome Project was
to determine the sequence of the three billion base
pairs that make up the human genome and to identify
its approximately 25,000 genes from physical and func-
tional standpoints. Most of the human genome (95%)
had been sequenced by the end of 2003; of the
remainder it is likely that the centromeres and telo-
meres will remain unsequenced until suitable new
technology is developed. Current research efforts are
Molecular Pathology # 2009, Elsevier, Inc. All Rights Reserved.
n Manel Esteller
directed toward analyzing the evolution of the
genome, differences between individuals (polymorph-
isms), and environmental effects. The near-complete
sequence has provided a rich source of scientific
knowledge and has initiated new fields of genomic
applications. However, the sequence itself does not
predict how the genome is packaged in chromatin to
ensure the differential expression of genes, which is
essential for development and differentiation [1]. Epi-
genetic processes, defined as the heritable patterns of
gene expression that do not involve changes in the
sequence of the genome, and their effects on gene
activation and inactivation are increasingly understood
to be such a way of modulating phenotype transmis-
sion and development. Epigenetics links the fields of
genetics and developmental biology and can explain,
at least in part, the biological process by which identi-
cal genotypes establish patterns of differential gene
expression that are stable through cell division.
The Human Epigenome Project
To date, small-scale studies of specific epigenetic
marks have provided limited information about the
regulation of genes from different pathways, for exam-
ple, the hypermethylation-dependent silencing of
tumor suppressor genes in cancer or the mutational
inactivation of MeCP2 in Rett patients. However, we
need to develop an understanding of these processes
that is based on a broader perspective. A range of mat-
ters remains to be resolved, such as the relationships
between the epigenetic players (the epigenetic code)
and how the environment and/or aging modulate
the epigenetic marks. Much of this could be achieved
151
Part II Concepts in Molecular Biology and Genetics
by analyzing the epigenetic patterns on a genome-wide
scale, an approach that has at last become possible
thanks to recent technological advances. For example,
comprehensive DNA-methylation maps (called the
methylome) could be assessed by combining the
methyl-DIP strategy with tilling or promoter microarray
analyses [2–4]. In a similar manner, the use of the
chromatin immunoprecipitation technique followed
by genomic microarray hybridization (ChIP-on-chip)
has begun to provide extensive maps of histone modifi-
cations [5–7]. Although the groundbreaking discov-
eries in the field of human disease were initially
performed in cancer cells, the characterization of the
error-bearing epigenomes underlying other disorders,
such as neurological, cardiovascular, and immunologi-
cal pathologies, has only just begun [8]. The fact that
epigenetic aberrations control the function of the
human genome and contribute to normal and patho-
logical states justifies carrying out a comprehensive
human epigenome project. The goal of the Human
Epigenome Project is “to identify all the chemical
changes and relationships among chromatin constitu-
ents that provide function to the DNA code.” This “will
allow a fuller understanding of normal development,
aging, abnormal gene control in cancer, and other dis-
eases as well as the role of the environment in human
health” [1]. It is important to bear in mind that there
is no single epigenome, but rather many different ones
that are characteristic of normal and diverse human dis-
orders, so it is essential to define the chosen starting
material [1]. The information extracted from the
whole-genome assays will help us understand the role
of the epigenetic marks, and could have translational
research benefits for diagnosis, prognostic, and thera-
peutic treatment [8]. Defining human epigenomes
associated with human disorders will help select patients
who are likely to benefit from epigenomic therapies or
prevention strategies, determine their efficacy and spec-
ificity, and lead to the identification of surrogate mar-
kers and end-points of its effects. The aim of the
present review is to provide an overview of how epige-
netic factors contribute to the development of human
diseases such as abnormal imprinting-causative patholo-
gies, cancer malignancies, as well as autoimmune, car-
diovascular, and neurological disorders with aberrant
epigenetic profiles.
GENOMIC IMPRINTING
Epigenetic Regulation of Imprinted Genes
Genomic imprinting is a genetic phenomenon by
which epigenetic chromosomal modifications drive
differential gene expression according to the parent-
of-origin. Expression is exclusively due either to the
allele inherited from the mother (such as the H19
and CDKN1C genes) or to that inherited from the
father (such as IGF2). It is an inheritance process that
is independent of the classical Mendelian model. Most
imprinted genes, which have been identified in
insects, mammals, and flowering plants, are involved
in the establishment and maintenance of particular
152
phases of development. Nucleus transplantation experi-
ments in mouse zygotes carrying reciprocal transloca-
tions carried out in the early 1980s suggested that
imprinting may be fundamental to mammalian devel-
opment [9]. Assays confirmed that normal gene
expression and development in mice require the con-
tribution of both maternal and paternal alleles. How-
ever, it was not until 1991 that the first imprinted
genes, insulin-like growth factor 2 (IGF2) and its
receptor (IGF2R), were identified [10]. Since then,
83 imprinted genes have been identified in mice and
humans, about one-third of which are imprinted in
both species [11]. It has recently been predicted that
600 genes have a high probability of being imprinted
in the mouse genome, and a similar genome-wide
analysis predicts humans to have about half as many
imprinted genes [12]. The molecular mechanisms
underlying genomic imprinting are poorly under-
stood. As imprinting is a dynamic process and the pro-
file of imprinted genes varies during development,
regulation must be epigenetic. DNA methylation has
been widely described as the major mechanism
involved in the control of genes subjected to imprint-
ing. One model for this regulation is based on the
cluster organization of imprinted genes. This structure
within clusters allows them to share common regu-
latory elements, such as noncoding RNAs and differ-
entially methylated regions (DMRs). DMRs are up to
several kilobases in size, rich in CpG dinucleotides
(such as CpG islands), and may contain repetitive
sequences. DNA methylation of DMRs is thought
to interact with histone modifications and other chro-
matin proteins to regulate parental allele-specific
expression of imprinted genes. Furthermore, the
aforementioned regulatory elements usually control
the imprinting of more than one gene, giving rise to
imprinting control regions (ICRs). This cluster organi-
zation, observed in 80% of imprinted genes, and the
specific DNA methylation patterns associated with
DMRs are two of the main characteristics of imprinted
genes. Deletions or aberrations in DNA methylation of
ICRs lead to loss of imprinting (LOI) and inappropri-
ate parental gene expression [13]. Imprinted genes
have diverse roles in growth and cellular proliferation,
and specific patterns of genomic imprinting are estab-
lished in somatic and germline cells [12,14]. Imprint-
ing is erased in germline cells, and reprogramming
involving a de novo methyltransferase is necessary to
ensure sex-specific gene expression in the individual.
Methyl groups are incorporated into most ICRs in
oocytes, although only some ICRs are methylated dur-
ing spermatogenesis. After fertilization, the specific
methylation profiles of ICRs must be maintained dur-
ing development in order to mediate the allelic expres-
sion of imprinted genes. In the primordial germline
cells of the developed individual, these imprinting
marks must be freshly erased by DNA demethylation
to allow the subsequent establishment of new oocyte-
specific and sperm-specific imprints. As a consequence,
mammalian imprinting can be described as a develop-
ment-dependent cycle based on germline establish-
ment, somatic maintenance, and erasure.
 Chapter 8 The Human Epigenome: Implications for the Understanding of Human Disease
Imprinted Genes and Human
Genetic Diseases
Since expression of imprinted genes is monoallelic, and
thereby functionally haploid, there is no protection from
recessive mutations that the normal diploid genetic com-
plement would provide [12]. For this reason, genetic and
epigenetic aberrations in imprinted genes are linked to a
wide range of diseases [15]. Modulation of perinatal
growth and human pregnancy has played a central role
in the evolution of imprinting, and many of the diseases
associated with imprinted genes involve some disorders
of embryogenesis. This is the case of the hydatidiform
mole disorder, where all nuclear genes are inherited
from the father. In most cases, this androgenesis arises
when an anuclear egg is fertilized by a single sperm, after
which all the chromosomes and genes are duplicated.
However, fertilization by two haploid sperm (diandric
diploidy) may occasionally occur. Most cases are sporadic
and androgenetic, but recurrent hydatidiform mole has
biparental inheritance with disrupted DNA methylation
of DMRs at imprinted loci [16]. In contrast, the disorder
of ovarian dermoid cysts arises from the spontaneous acti-
vation of an ovarian oocyte that leads to the duplication
of the maternal genome [17]. These abnormalities sug-
gest that normal human development is possible only
when the paternal and maternal genomes are correctly
transmitted. Parent-of-origin effects involved in behav-
ioral and brain disorders have been widely reported at
the prenatal and postnatal stages of development [18].
A postnatal growth retardation syndrome associated with
the MEST gene expression is an illustrative example. The
effect of introducing a targeted deletion into the coding
sequence of the mouse MEST gene strongly depends on
the paternal allele [19]. When the deletion is paternally
derived, Mestþ/– mice are viable and fertile, but mutant
mice show growth retardation and high mortality.
Mest–/þ animals, with a maternally derived deletion,
show none of these effects. This suggests that the pheno-
typic consequences of this mutation are detected only
through paternal inheritance and are the result of
imprinting. There is also evidence that some imprinting
effects are associated with increased susceptibility to can-
cer. Absence of expression of a tumor suppressor gene
could be the result of LOI or uniparental disomy
(UPD) in imprinted genes. Conversely, LOI or UPD of
an imprinted gene that promotes cell proliferation (an
oncogene) may allow gene expression to be inappropri-
ately increased. These aberrations could have a wide-
spread effect if the aberrant imprinting occurs in an
ICR, resulting in the epigenetic dysregulation of multiple
imprinted oncogenes and/or tumor suppressor genes
[20]. The most common genetic disorders associated
with imprinting aberrations are described next.
Prader-Willi Syndrome and
Angelman Syndrome
Prader-Willi syndrome (PWS; OMIM #176270) and Angel-
man syndrome (AS; OMIM #105830) are very rare genetic
disorders with autosomal dominant inheritance in which
gene expression depends on parental origin. The clinical
features of both syndromes are quite similar; they are neu-
rological disorders with mental retardation and develop-
mental aberrations [21]. PWS is characterized by
diminished fetal activity, feeding difficulties, obesity, mus-
cular hypotonia, mental retardation, poor physical coordi-
nation, short stature, hypogonadism, and small hands and
feet, amongst other traits. AS is characterized by mental
retardation, movement or balance disorder, characteristic
abnormal behaviors, increased sensitivity to heat, absent
or little speech, and epilepsy. The prevalence of the two
syndromes is not accurately known, but is estimated to
be between 1 in 12,000 and 1 in 15,000 live births, respec-
tively. Most cases of PWS are caused by the deficiency of
the paternal copies of the imprinted genes on chromo-
some 15 located in the 15q11–q13 region, while AS affects
maternally imprinted genes in the same region. This defi-
ciency could be due to the deletion of the 15q11–q13
region (3–4 Mb), parental uniparental disomy of chromo-
some 15, or imprinting defects [22]. The imprinted
domain on human chromosome 15q11–q13 is regulated
by an ICR that is responsible for establishing the imprint-
ing in the gametes and for maintaining the patterns dur-
ing the embryonic phases. ICR regulates differential
DNA methylation and chromatin structure, and in conse-
quence, differential gene expression affecting the two
parental alleles. The ICR in 15q11–q13 appears to have a
bipartite structure; one part seems to be responsible for
the control of paternal expression and the other for
maternal gene expression [23]. PWS is in effect a contigu-
ous gene syndrome resulting from deficiency of the pater-
nal copies of the imprinted SNRF/SNRPN gene, the
necdin gene, and possibly other genes [24,25]. It has been
estimated that the region could contain more than 30
genes, so PWS probably results from a stochastic partial
inactivation of important genes [26]. While PWS appears
to be more closely related to deficiencies caused by chro-
matin aberrations affecting several genes, AS is associated
with mutations in single genes. For instance, the most
common genetic defect leading to AS is a 4 Mb maternal
deletion in chromosomal region 15q11–13, which causes
an absence of UBE3A expression in the maternally
imprinted brain regions. Mutations in the gene encoding
the ubiquitin-protein ligase E3A (UBE3A) have been iden-
tified in 25% of AS patients [27]. The UBE3A gene is pres-
ent on both the maternal and paternal chromosomes,
but differs in its pattern of methylation [28]. Paternal
silencing of the UBE3A gene occurs in a brain-region-spe-
cific manner, with the maternal allele being active almost
exclusively in the Purkinje cells, hippocampus, and cere-
bellum [29]. Another maternally expressed gene,
ATP10C (aminophospholipid-transporting ATPase), is
also located in this region and has been implicated in
AS [30]. Like UBE3A, it exhibits imprinted, preferential
maternal expression in human brain [31].
Beckwith-Wiedemann Syndrome
Beckwith-Wiedemann syndrome (BWS; OMIM #130650)
is a well-characterized human disease involving imprin-
ted genes that are epigenetically regulated, and studies
153
Part II Concepts in Molecular Biology and Genetics
of BWS patients have contributed much to the under-
standing of normal imprinting. BWS is a rare genetic
or epigenetic overgrowth syndrome with an estimated
prevalence of about 1 in 15,000 births and a high mor-
tality rate in the newborn (about 20%). Neonatal
patients are mainly characterized by exomphalos,
macroglossia, and gigantism, but other symptoms may
occur, such as organomegaly, adrenocortical cytome-
galy, hemihypertrophy, and neonatal hypoglycemia
[32]. There is also an increased risk of developing spe-
cific tumors, such as Wilms’ tumor and hepatoblastoma
[33]. The imprinted domain BWS-related genes are
located on 11p15 and are regulated by a bipartite
ICR. Two clusters of imprinting genes have been
described [34]: (i) H19/IGF2 (imprinted, maternally
expressed, untranslated mRNA/insulin-like growth fac-
tor 2); and (ii) p57KIP2 (a cyclin-dependent kinase
inhibitor), TSSC3 (a pleckstrin homology-like domain),
SLC22A1 (an organic cation transporter), KvLQT1 (a
voltage-gated potassium channel), and LIT1 (KCNQ1
overlapping transcript 1). Both clusters are regulated
by two DMRs differentially methylated regions: DMR1,
which is responsible for H19/IGF2 control and is
methylated on the paternal but not the maternal allele,
and DMR2, which is located upstream of LIT1 and
is normally methylated on the maternal but not the
paternal allele. BWS can appear as a consequence of
two separate mechanisms [35]. First, some patients
are characterized by UPD, which consists of the com-
plete genetic replacement of the maternal allele region
with a second paternal copy, and/or LOI affecting
the IGF2-containing region [36] and/or the LIT1 gene
[37], which causes a switch in the epigenotype of
the H19/IGF2 subdomain or the p57/ KvLQT1/LIT1
subdomain, respectively. When UPD affects IGF2, it
yields a double dose of this autocrine factor, resulting
in tissue overgrowth and increased cancer risk [38].
The LOI mechanism involves aberrant methylation of
the maternal H19 DMR [39]. Second, maternal repla-
cements of the allele and localized abnormalities of
allele-specific chromatin modification on p57KIP2
(also known as CDKN1C) [40] or LIT1 could also con-
tribute to the BWS phenotype [41]. In conclusion,
BWS is a model for the hierarchical organization of epi-
genetic regulation in progressively larger domains
[42]. Additionally, mutations in NSD1 (nuclear receptor-
binding SET domain-containing protein 1), the major
cause of the Sotos overgrowth syndrome, have been
described in BWS patients, demonstrating the role this
gene plays in imprinting the chromosome 11p15
region [43].
CANCER EPIGENETICS
Cancer encompasses a fundamentally heterogeneous
group of disorders affecting different biological pro-
cesses, and is caused by abnormal gene/pathway func-
tion arising from specific alterations in the genome.
Initially, cancer was thought to be solely a consequence
of genetic changes in key tumor suppressor genes and
oncogenes that regulate cell proliferation, DNA repair,
154
cell differentiation, and other homeostatic functions.
However, recent research suggests that these altera-
tions could also be due to epigenetic disruption. The
study of epigenetic mechanisms in cancer, such as
DNA methylation, histone modification, nucleosome
positioning and microRNA expression, has provided
extensive information about the mechanisms that con-
tribute to the neoplastic phenotype through the regu-
lation of expression of genes critical to transformation
pathways. These alterations and their involvement in
tumor development are briefly reviewed in the follow-
ing sections.
DNA Hypomethylation in Cancer Cells
The low level of DNA methylation in tumors compared
with that in their normal-tissue counterparts was one
of the first epigenetic alterations to be found in
human cancer [44]. It has been estimated that 3–6%
of all cytosines are methylated in normal human
DNA, although cancer cell genomes are usually hypo-
methylated with malignant cells, featuring 20–60% less
genomic 5-methylcytosine than their normal counter-
parts [45]. Global hypomethylation in cancer cells
is generally due to decreased methylation in CpGs
dispersed throughout repetitive sequences, which
account for 20–30% of the human genome, as well as
in the coding regions and introns of genes [45]. A
chromosome-wide and large-promoter-specific study
of DNA methylation in a colorectal cancer cell line
using the methyl-DIP approach has revealed extensive
hypomethylated genomic regions located in gene-poor
areas [2]. Importantly, the degree of hypomethylation
of genomic DNA increases as the lesion progresses
from a benign cellular proliferation to an invasive
cancer [46]. From a functional point of view, hypo-
methylation in cancer cells is associated with a number
of adverse outcomes, including chromosome instab-
ility, activation of transposable elements, and LOI
(Figure 8.1). Decreased methylation of repetitive
sequences in the satellite DNA of the pericentric
region of chromosomes is associated with increased
chromosomal rearrangements, mitotic recombination,
and aneuploidy [47,48]. Intragenomic endoparasitic
DNA, such as L1 (long interspersed nuclear elements)
[49] and Alu (recombinogenic sequence) repeats, are
silenced in somatic cells and become reactivated in
human cancer. Deregulated transposons could cause
transcriptional deregulation, insertional mutations,
DNA breaks, and an increased frequency of recombi-
nation, contributing to genome disorganization,
expression changes, and chromosomal instability
[49]. DNA methylation underlies the control of several
imprinted genes, so the effect on the loss of imprint-
ing must also be considered. Wilms’ tumor, a nephro-
blastoma that typically occurs in children, is the best-
characterized imprinting effect associated with
increased susceptibility to cancer [50]. Other changes
in the expression of imprinted genes caused by
changes in methylation have been demonstrated in
malignancies such as osteosarcoma [51], hepatocellu-
lar carcinoma [52], and bladder cancer [53]. Finally,
 Chapter 8 The Human Epigenome: Implications for the Understanding of Human Disease

Promoter of TSGs Repetitive sequence

RNA HDAC
Pol MBD
TF
HMT
Me Me Rb
Ac Ac Ac Me
MeMe Ac
MeMe MeMe MeMe

K16H4 K8H4 K4H3 K16H4 K20H4 K9H3 K27H3

K5H4 K12H4

Normal Cell

Normal Cell
TRANSCRIPTION REPRESSION

Unmethylated CpG
Methylated CpG HDAC
MBD
HMT Me
Ac Ac Ac
Me MeMe MeMe
MeMe MeMe
K16H4 K8H4 K4H3 K20H4
K9H3 K27H3 K5H4 K12H4
Cancer Cell

Cancer Cell
A SILENCING B ACTIVATION

Figure 8.1 A model for the disruption of histone modifications and DNA methylation patterns in cancer cells.
Nucleosome arrays are located in the context of genomic regions that include (A) promoters of tumor suppressor genes
(TSGs), (B) repetitive sequences in heterochromatin regions. Nucleosomes consisting of two copies of histones H2A, H2B,
H3, and H4 are represented as gray cylinders. DNA (black lines) is wrapped around each nucleosome. In normal cells,
TSG promoter regions are unmethylated and enriched in histone modification marks associated with active transcription,
such as acetylation of histone H4 (at lysine K5, K8, K12, and K16), or trimethylation of histone H3 (at lysine K4).
Transcription machinery recognizes these active marks and transcription of TSGs is allowed. In the same cells, the
repetitive genomic DNA is silenced due to the high degree of DNA methylation and histone-repressive marks: histone H4 is
densely trimethylated at lysine 20, and histone H3 is dimethylated at lysine 9 and trimethylated at lysine k27. This
epigenetic profile is disrupted in transformed cells. TSG promoters are silenced by the loss of the histone-active marks and
gain of promoter hypermethylation. Repetitive sequences are activated by replacement of the repressive marks, leading to
activation of endoparasitic sequences, genomic instability, or loss of imprinting.

DNA methylation acts a mechanism for controlling a hypomethylation-dependent manner. Activation of

cellular differentiation, allowing the expression only PAX2, a gene that encodes a transcription factor
of tissue-specific and housekeeping genes in somatic involved in proliferation and other important cell
differentiated cells. It is possible that some tissue- activities, and let-7a-3, an miRNA gene, have been
specific genes became reactivated in cancer [54] in implicated in endometrial and colon cancer [55,56].

155
Part II Concepts in Molecular Biology and Genetics
Hypermethylation of Tumor
Suppressor Genes
Aberrations in DNA methylation patterns of the CpG
islands in the promoter regions of tumor suppressor
genes are accepted as being a common feature of
human cancer (Figure 8.1). The initial discovery of
silencing was performed in the promoter of the retino-
blastoma (Rb) tumor suppressor gene [57], but hyper-
methylation of genes like VHL (associated with von
Hippel-Lindau disease), p16INK4a, 8-11 hMLH1 (a
homologue of Escherichia coli MutL), and BRCA1
(breast-cancer susceptibility gene 1) has also been
described [58]. The presence of CpG island promoter
hypermethylation affects genes from a wide range of
cellular pathways, such as cell cycle, DNA repair, toxic
catabolism, cell adherence, apoptosis, and angiogene-
sis, among others [45], and may occur at various stages
in the development of cancer. In recent years, a CpG-
island hypermethylation profile of human primary
tumors has emerged, which shows that the CpG island
hypermethylation profiles of tumor suppressor genes
are specific to the cancer type [59,60]. Each tumor
type can be assigned a specific, defining DNA “hyper-
methylome,” rather like a physiological or cytogenetic
marker. These marks of epigenetic inactivation occur
not only in sporadic tumors but also in inherited cancer
syndromes, in which hypermethylation may be the sec-
ond lesion in Knudson’s two-hit model of cancer devel-
opment [61]. It is expected that improvements in
genome-wide epigenomic studies will increase the num-
ber of hypermethylated tumor suppressor genes in a
broad spectrum of tumors. However, to date 100–400
instances of gene-specific methylation have been noted
in a given tumor. Sometimes the epigenetic alteration
of a tumor suppressor gene has genetic consequences,
for example, when a DNA repair gene (hMLH1, BRCA1,
MGMT, or Werner’s syndrome gene) is silenced by pro-
moter methylation and functionally blocked [58,62].
Histone Modifications of Cancer Cells
The histone modification network is very complex.
Histone modifications can occur in various histone pro-
teins (including H2B, H3, H4) and variants (such as
H3.3) and affect different histone residues (including
lysine, arginine, serine). Several chemical groups
(methyl, acetyl, phosphate) may be added in different
degrees (for example, monomethylation, dimethylation,
or trimethylation). Furthermore, the significance of
each modification depends on the organism, the
biological process, and the chromatin-genomic region.
Due to this diversity of permutations and combinations,
little is known about the patterns of histone modification
disruption in human tumors. Recent results have shown
that the CpG promoter-hypermethylation event in
tumor suppressor genes in cancer cells is associated with
a particular combination of histone markers: deacetyla-
tion of histones H3 and H4, loss of H3K4 trimethylation,
and gain of H3K9 methylation and H3K27 trimethyla-
tion (Figure 8.1) [63]. Increased acetylated histones H3
and H4, and H3/K4 at the p21 and p16 transcription start
156
sites after genistein induction (an isoflavone found in
the soybean with tumor suppressor properties), in the
absence of p21 promoter methylation, have been
reported [64]. The association between DNA methyla-
tion and histone modification aberrations in cancer also
occurs at the global level. In human and mouse tumors,
histone H4 undergoes a loss of monoacetylated and tri-
methylated lysines 16 and 20, respectively, especially in
the repetitive DNA sequences [46]. Subsequent studies
showed that loss of trimethylation at H4K20 is involved
in disrupting heterochromatic domains and may reduce
the response to DNA damage of cancer cells [65]. Immu-
nohistochemical staining of primary prostatectomy tis-
sue samples revealed that patterns of H3 and H4 were
predictors of clinical outcome independently of tumor
stage, preoperative prostate-specific antigen levels, and
capsule invasion in prostate cancer [66].
Epigenetic Regulation of microRNAs
in Cancer
miRNA expression patterns must be tightly regulated
during development in a tissue-specific manner, and
play important roles in cell proliferation, apoptosis,
and differentiation [67]. miRNA expression profiles dif-
fer between normal and tumor tissues, and also among
tumor types [68], whereby some microRNAs are down-
regulated in cancer (like tumor suppressor genes). This
comparison suggests that miRNAs could be silenced by
epigenetic mechanisms [69]. DNA methylation has
been shown to be the regulatory mechanism in at least
two microRNAs, miR-127 and miR-124a [69]. miR-127,
which negatively regulates the protooncogene BCL6
(B-Cell Lymphoma 6), is usually expressed in normal
cells but is silenced by DNA methylation in cancer cells
[70]. Similarly, the hypermethylation-dependent silenc-
ing of miR-124a results in an increase in expression of
cyclin D-kinase 6 oncogene (CDK6), and is recognized
as a common feature of a wide range of tumors [71].
Aberrations in Histone-Modifier Enzymes
Aberrations in the epigenetic profiles, with respect to
DNA methylation and histone modifications, could also
be a consequence of genetic disruption of the epige-
netic machinery. A preliminary set of genes involved
in epigenetic modifications with mutations in cancer
cells but not in the normal counterparts has been
found[45]. A list of genes involved in epigenetic modi-
fications that are disrupted in human cancer is pre-
sented in Table 8.1. Although alterations in the levels
of DNMTs and MBD-containing proteins are com-
monly observed in human tumors [72,73], no genetic
lesion has been described in the DNA-methylation
machinery in cancer cells. The picture is different for
histone modifier enzymes. In leukemia and sarcoma,
chromosomal translocations that involve histone-modi-
fier genes, such as histone acetyltransferases (HATs)
[74] [such as cyclic AMP response-element-binding
protein (CREB)-binding protein-monocytic leukemia
zinc finger (CBP-MOZ)] and histone methyltransferases
(HMTs) [such as mixed-lineage leukemia 1 (MLL1)]
 Chapter 8 The Human Epigenome: Implications for the Understanding of Human Disease

Table 8.1 Disruption of Genes Involved in DNA Methylation and Histone Modifications in Cancer

Gene Alteration Tumor type

Alterations affecting DNA methylation enzymes (DNMTs)

DNMT1 Overexpression Various
DNMT3b Overexpression Various
Alterations involving Methyl-CpG-binding proteins (MBPs)
MeCP2 Overexpression, rare mutations Various
MBD1 Overexpression, rare mutations Various
MBD2 Overexpression, rare mutations Various
MBD3 Overexpression, rare mutations Various
MBD4 Mutations in microsatellite instable tumors Colon, stomach, endometrium
Alterations disrupting histone acetyltransferases (HATs)
p300 Mutations in microsatellite instable tumors Colon, stomach, endometrium
CBP Mutations, translocations, deletions Colon, stomach, endometrium, lung, leukemia
pCAF Rare mutations Colon
MOZ Translocations Hematological malignancies
MORF Translocations Hematological malignancies, leiomyomata
Alterations disrupting histone deacetylases (HDACs)
HDAC1 Aberrant expression Various
HDAC2 Aberrant expression, mutations in microsatellite instable Various
tumors
Alterations affecting histone methyltransferases (HMTs)
MLL1 Translocation Hematological malignancies
MLL2 Gene amplification Glioma, pancreas
MLL3 Deletion Leukemia
NSD1 Translocation Leukemia
EZH2 Gene amplification, overexpression Various
RIZ1 Promoter CpG-island hypermethylation Various
Alterations affecting histone demethylases
GASC1 Gene amplification Squamous cell carcinoma

Adapted from [45].

[75], nuclear-receptor binding SET domain protein 1
(NSD1) [76], and nuclear-receptor binding SET-
domain protein 3 (NSD3) [77], create aberrant fusion
proteins [8]. In solid tumors, both HMT genes such as
EZH2 [78], mixed-lineage leukemia 2 (MLL2) [79], or
NSD3 [80], and a demethylase [Jumonji domain-con-
taining protein 2C (JMJD2C/GASC1)] are known to be
amplified [81]. Genetic aberrations also disrupt expres-
sion of histone deacetylases, such as histone deacetylase
2 (HDAC2), which could be affected by mutational
frameshift inactivation in colon cancer [82], and chro-
matin remodeling proteins, such as HLTF (helicase-like
transcription factor) [83], BRG1 (Brahma-related gene
1) [84], and other components of the SWI/SNF family
of proteins.
HUMAN DISORDERS ASSOCIATED
WITH EPIGENETICS
Aberrant Epigenetic Profiles Underlying
Immunological, Cardiovascular,
Neurological, and Metabolic Disorders
The patterns of DNA methylation, histone modifications,
microRNAs, and several chromatin-related proteins of
sick cells usually differ from those of healthy cells, high-
lighting the importance of epigenetic regulation in most
human pathologies. Most of our knowledge regarding
human epigenetic diseases was first obtained from cancer
cells, but nowadays there is increasing interest in under-
standing the role of epigenetic modifications in the etiol-
ogy of human disease. This section summarizes the
involvement of specific epigenetic aberrations in immu-
nological, cardiovascular, and neurological disorders.
DNA methylation is the best-characterized epigenetic
modification in many pathways of immunology, and is
the source of much of our knowledge about the molecu-
lar network of the immune system. Classical autoimmune
disorders, such as systemic lupus erythematosus (SLE),
an autoimmune disease characterized by the production
of a variety of antibodies against nuclear components and
which causes inflammation and injury of multiple
organs, and rheumatoid arthritis, a chronic systemic
autoimmune disorder which primarily causes inflamma-
tion and destruction of the joints, are characterized
by massive genomic hypomethylation [85,86]. This
decrease in DNA methylation levels is highly reminiscent
of the global demethylation observed in the DNA of
tumor cells compared with their normal tissue counter-
parts [8]. How this hypomethylation in T-cells induces
SLE is not well understood. It has been proposed that
demethylation induces overexpression of integrin adhe-
sive receptors and leads to an autoreactive response.
Identification of the full set of genes deregulated by
DNA hypomethylation could help to explain these
immunological disorders and will also enable the devel-
opment of effective therapies to cure SLE [85]. More
pathologies with epigenetic regulation of immunology
have been reported, including the epigenetic silencing
157
Part II Concepts in Molecular Biology and Genetics
of the ABO histo-blood group genes [87], the silencing of
human leukocyte antigen (HLA) class I antigens [88],
and the melanoma antigen-encoding gene (MAGE) fam-
ily. It is known that MAGE gene expression is epigeneti-
cally repressed by promoter CpG methylation in most
cells, but MAGE genes may be expressed in various tumor
types via CpG demethylation and can act as antigens that
are recognized by cytolytic T-lymphocytes [89]. Altera-
tions of specific genomic DNA methylation levels have
been described not only in the fields of oncology and
immunology but also in a wide range of biomedical and
scientific fields. In neurology, for example, mutations in
MeCP2, which encodes methyl CpG binding protein 2,
cause most cases of Rett syndrome (RTT) and autism
[90]. Future research needs to determine whether aber-
rant DNA methylation is important in the complex etiol-
ogy of other frequent neurological pathologies, such as
schizophrenia and Alzheimer’s disease. Beyond this,
DNA methylation changes are also known to be involved
in cardiovascular disease, the biggest killer in Western
countries [8]. For example, aberrant CpG island hyper-
methylation has been described in atherosclerotic lesions
[91]. Since DNA methylation and histone modifications
are mechanistically linked, it is likely that different
changes in DNA methylation are associated with changes
in histone modifications in human diseases. To date, we
have been largely ignorant of how these histone modifi-
cation markers are disrupted in human diseases. A pre-
view of the patterns of histone modifications and their
cellular location has only been described for cancer
malignancies [45]. For other human pathologies, our
knowledge of the alterations in histone modification
patterns comes from the use of epigenetic drugs. Thera-
peutic assays have demonstrated that histone deacetylase
(HDAC) inhibitors can improve deficits in synaptic plas-
ticity, cognition, and stress-related behaviors in a wide
range of neurological and psychiatric disorders, includ-
ing Huntington’s and Parkinson’s diseases, anxiety and
mood disorders, and Rubinstein-Taybi and Rett syn-
dromes [92]. Abnormal histone modification patterns
associated with specific gene expression have also been
described in lupus CD4þ T-cells [93], and HDAC inhibi-
tors are able to reverse gene expression significantly [85].
Genetic Aberrations Involving Epigenetic
Genes
Genetic alterations of genes coding for enzymes that
mediate chromatin structure could result in a loss of ade-
quate regulation of chromatin compaction, and finally,
the deregulation of gene transcription and inappropriate
protein expression. Although the consequences in cancer
malignancies have been widely described, in this section
we extend the review to include other genetic diseases
involving the function of several enzymes of the epigenetic
machinery. The phenotype of these diseases also helps to
clarify the role of various chromatin proteins in cell prolif-
eration and differentiation. These include disorders aris-
ing from alterations in chromatin remodeling factors,
alterations of the components of the DNA methylation
machinery, and aberrations disturbing histone modifiers.
158
Syndromes of Disordered Chromatin Remodeling
Alpha-Thalassemia X-Linked Mental Retardation
Syndrome Alpha-thalassemia X-linked mental retar-
dation (ATRX; OMIM #301040) syndrome is charac-
terized by distinctive craniofacial features, genital
anomalies, and severe developmental delays with hypo-
tonia and mental retardation. Some individuals with
ATRX syndrome have an unusual mild form of hemo-
globin H disease, but alpha-thalassemia is not a constant
characteristic of the syndrome [94]. ATRX is the gene
associated with ATRX syndrome and codes for a cen-
tromeric heterochromatin-binding protein from the
SNF2 family of helicase/ATPases. Centromeric ATRX
is required for maintaining a bipolar metaphase II spin-
dle and for establishing correct chromosome alignment
during meiosis. Its activity has been associated with
epigenetic modifications, such as histone deacetylation
and DNA methylation [95].
CHARGE Syndrome The acronym CHARGE (OMIN
#214800) stands for Coloboma of the eye, Heart
anomaly, choanal Atresia, Retardation of mental and
somatic development, Genital and/or urinary abno-
rmalities, and Ear abnormalities and/or deafness
[96]. The estimated incidence of CHARGE syndrome
is 1 in 8,500–12,000 births. It is an autosomal domi-
nant condition with genotypic heterogeneity. Most
cases are due to the mutation or deletion of a mem-
ber of the chromodomain helicase DNA-binding
domain (CHD7) family of ATP-dependent chromatin-
remodeling enzymes [97], and in consequence, a pro-
tein involved in chromatin structure and gene
expression.
Cockayne Syndrome Patients with Cockayne syndrome
type B (CSB; OMIM #133540) present with failure to
thrive, short stature, premature aging, neurological
alterations, photosensitivity, delayed eruption of the
primary teeth, congenital absence of some permanent
teeth, partial macrodontia, atrophy of the alveolar
process, and caries [98]. CSB is a genetic disorder with
autosomal recessive inheritance that is caused by muta-
tion of the excision-repair cross-complementing group
6 (ERCC6) gene. The protein, with ATP-stimulated
ATPase activity, is part of the nucleotide excision repair
(NER) pathway, a complex system that eliminates a
broad spectrum of structural DNA lesions, including
ultraviolet (UV)-induced dimers and DNA cross-links
[99].
Schimke Immunoosseous Dysplasia Schimke immu-
noosseous dysplasia (SIOD; OMIM #242900) is an
autosomal recessive disorder with the diagnostic
features of growth retardation, renal failure, recur-
rent infections, cerebral infarcts, slowly progressive
immune defects, such as T-cell immunodeficiency
and skin pigmentation, that begin in childhood
[100]. Mutations in a component of the SWI/SNF
complex, the SMARCAL1 gene, are responsible for
this condition [101].
 Chapter 8 The Human Epigenome: Implications for the Understanding of Human Disease
DNA Methylation-Associated Diseases
Immunodeficiency-Centromeric Instability-Facial
Anomalies Syndrome
Immunodeficiency-centromeric instability-facial ano-
malies syndrome (ICF syndrome; OMIM #242860) is
characterized by immunodeficiency in association with
centromere instability of chromosomes 1, 9, and 16,
and facial anomalies. ICF syndrome patients exhibit
facial anomalies, such as hypertelorism, low-set ears,
epicanthal folds, and macroglossia. It also features
variable reductions in serum immunoglobulin levels
that cause most ICF syndrome patients to succumb to
infectious diseases before adulthood [102]. It is a rare
autosomal recessive disorder that is notable for the
variable severity of chromosomal abnormalities. Essen-
tially, these involve juxtacentromeric heterochromatin
formation of chromosomes 1, 9, and 16; an increased
frequency of somatic recombination between the arms
of these chromosomes; and a marked tendency to form
multibranched configurations [103]. The observation
that the cytogenetic abnormalities in the phenotype of
ICF syndrome could be compared with the decondensa-
tion effect found in a normal pro-B lymphoblastoid
cell line treated with the DNA methylation inhibitors
5-azacytidine or 5-azadeoxycytidine suggested the involve-
ment of genomic DNA methylation in this disorder.
In fact, 40% of ICF syndrome patients owe their condi-
tion to mutations in the DNMT3B gene, which encodes
a de novo DNA-methyltransferase that acts on CpG-rich
satellite DNAs [104]. In this way, ICF syndrome can be
biochemically characterized by hypomethylation of
CpG sites in some heterochromatic regions.
Rett Syndrome
Rett syndrome (RTT; OMIM #312750) is a severe neuro-
developmental disorder that primarily occurs in females
at an estimated frequency of 1 in 15,000 births. It is char-
acterized by arrested development between 6 and 18
months of age, regression of acquired skills, loss of
speech, stereotypical movements (classically of the
hands), microcephaly, seizures, and mental retardation.
Rarely, RTT has also been described in male patients
with somatic mosaicism or an extra X chromosome
[105]. Mutations in the coding regions of the MeCP2
(methyl CpG-binding protein 2) gene are found in
approximately 70–80% of classic RTT patients [106],
while the remaining cases result from large deletions
of the MeCP2 gene [107], mutations in noncoding
regions of the MeCP2 gene, and mutations in other
genes [108]. An atypical Rett syndrome with infantile
spasms that may be related to mutations in the cyclin-
dependent kinase-like 5 (CDKL5) gene has also been
described [109]. The MeCP2 gene encodes a protein
that preferentially binds methylated CpG dinucleotides
and, in turn, mediates transcriptional repression
through the recruitment of histone deacetylases, his-
tone methyltransferases, and polycomb proteins,
among others [110]. In addition, cells of RTT patients
may have an abnormal secondary chromatin structure,
with putative broad-ranging effects on expression of
genes that otherwise are not mutated [111]. It has been
proposed that MeCP2 disruption in RTT patients causes
aberrant upregulation of the genes that should nor-
mally be silenced in the nervous system, like the BDNF
gene or imprinted genes [112]. The discovery that
MeCP2 was the gene responsible for RTT syndrome
allows it to be used for early diagnosis and prenatal
detection. In addition, the finding that epigenetic regu-
lation has a role in the pathogenesis of RTT has sug-
gested opportunities for therapy.
Human Malignancies Associated with
Alterations in Histone Modifications
Coffin-Lowry Syndrome
The symptoms of Coffin-Lowry syndrome (CLS; OMIM
#303600) are usually more severe in males than in
females since it is an X-linked condition. Males with
CLS syndrome usually exhibit severe-to-profound
mental retardation and delayed development, but in
female patients this may be entirely absent, or mild-to-
profound [113]. Other features, such as skeletal malfor-
mations, distinctive facial features, microcephaly, and
soft hands with short-tapered fingers, may be observed.
CLS has an estimated incidence of 1 in 40,000–50,000
births. It is caused by mutations in the RSK2 (ribosomal
protein S6 kinase) gene [114]. RSKs are involved in
activating the mitogen-activated kinase cascade and in
stimulating cell proliferation (transition between the
G0 and G1 phases of the cell cycle), and differentiation.
RSK2 is responsible for the phosphorylation of the tran-
scription factor CREB (cyclic AMP-responsive-binding
protein) and histone H3 as a mechanism for respond-
ing to mitogenic stimulation by epidermal growth
factor (EGF) [115]. Analysis of mRNA and protein
expression of RSK2 on lymphocytes extracted directly
from blood of patients revealed a mutation at a putative
phosphorylation site that would be critical for RSK2
activity. Both assays are rapid and practical techniques
for the diagnosis of CLS [116].
Rubinstein-Taybi Syndrome
Rubinstein-Taybi syndrome (RSTS; OMIM #180849) is
an autosomal dominant disorder characterized by
mental retardation, broad thumbs and toes, distinctive
facial abnormalities, and short stature. RSTS patients
have an increased risk of developing tumors, especially
congenital glaucoma. The incidence has been esti-
mated to be in 1 in 100,000 births [117]. In almost
all cases, the syndrome is caused by mutation of the
gene encoding the transcriptional coactivator CREB-
binding protein (CREBBP) [118]. Mutations in the
EP300 gene are responsible for a small percentage of
RSTS cases [119]. Both CREBBP and EP300 are his-
tone acetyltransferases. A direct connection between
loss of acetyltransferase activity and RSTS has been
described [120], which indicates that the disorder is
caused by aberrant chromatin regulation.
159
Part II Concepts in Molecular Biology and Genetics
Sotos Syndrome
Sotos syndrome (OMIM #117550) is a well-known over-
growth syndrome with autosomal dominant inheritance
characterized by excessive growth during childhood,
advanced bone age, macrocephaly, characteristic facial
gestalt, and various degrees of developmental delay
[121]. It may also be associated with variable minor
features, including cardiac and renal anomalies,
seizures, and scoliosis. The risk of neoplasm formation
in Sotos patients has been estimated to be about 2–3%
[122], which is greater than in the general population.
The most frequent tumor types are leukemia and lym-
phoma, but others, such as neural crest tumors, small-
cell lung cancer, and sacrococcygeal teratomas have
been reported [123]. Sotos syndrome is caused by hap-
loinsufficiency of the nuclear receptor-binding SET
domain-containing protein 1 gene (NSD1). Several
mutations cause the loss of NSD1 function in 90% of
patients with Sotos syndrome [121], such as truncating
mutations, missense mutations, 5q35 macrodeletions
and microdeletions encompassing the NSD1 gene or
the t(5;11)(q35;p15.5) translocation, which fuses NSD1
to nucleoporin-98 (NUP98 ). NSD1 contains a SET
domain that confers intrinsic histone methyltransferase
activity that is specific to lysine 36 of histone H3
(H3K36) and lysine 20 of histone H4 (H4K20), epige-
netic marks with ascribed roles in chromatin structure
and gene function.
ENVIRONMENT AND THE EPIGENOME
Gene expression strongly depends on environmental
signals—the external and endogenous stimuli—that
make up the cellular context. Since epigenetic states
can potentially be reverted, these gene-environment
interactions could be integrated by epigenetic modifica-
tions of the genome [124]. Disease susceptibility also
could be modified by environmental factors that alter
the profile of DNA methylation and histone modifica-
tions in normal cells during prenatal and postnatal life.
Alterations in the epigenome, and subsequent differen-
tial gene expression, could be directly affected by diet,
xenobiotic chemicals, behavior, and exogenous stimuli,
such as doses of radiation [12]. Aberrant epigenetic pat-
terns could be a consequence of changes in the metabo-
lism of the groups of donor chemicals, such as methyl or
acetyl groups. Diets that are deficient in folate and
methionine, which are necessary for normal biosynthe-
sis of S-adenosylmethionine (SAM), the methyl donor
for methylcytosine, lead to DNA hypomethylation and
aberrant imprinting of insulin-like growth factor
2 (IGF2) [125]. Mutations in methylenetetrahydrofolate
reductase (MTHFR-677T allele), which is critical for
directing the folate pool to remethylate homocysteine
to methionine, reduce the levels of DNA methylation
responsible for the pathogenesis of diseases related to
neural tube defects [126], spina bifida [127], and colo-
rectal cancer [128]. Associations are also known
between deficiency of methionine synthase, a folate-
dependent and vitamin B(12)-dependent enzyme, and
160
megaloblastic anemia, hemolytic uremic syndrome, and
pulmonary hypertension, among others [129]. Exposure
to metals, such as arsenic [130], cadmium [131], lead
[132], nickel [133], and chromium [134], is linked to
changes in the expression of epigenetically controlled
genes via interactions with DNA-methylation-associated
enzymes, histone acetyltransferase, and histone deacety-
lase enzymes. Endocrine-active chemicals, like estrogenic
and anti-androgenic toxins, decrease male fertility by
manipulating DNA methylation levels [135]. Among
the behavioral factors, maternal grooming is an influen-
tial environmental factor affecting the methylation
state of the glucocortocoid receptor in the hippocampus
of rat pups [136]. Reproductive factors like assisted
reproductive technology act as an environmental modu-
lator of the epigenome, and are associated with the
hypomethylation noted in Beckwith-Wiedemann and
Angelman syndromes [137]. Studies performed with
genetically identical individuals are an excellent method
for understanding the environment-epigenome interac-
tion [138,139]. In this way, the epigenome of human
monozygotic twins could also vary because of differences
in environmental factors. Recently, it was noted that
although twins are epigenetically indistinguishable
during the early years of life, older monozygous twins
exhibit marked differences in their overall content and
genomic distribution of 5-methylcytosine DNA and
histone acetylation, which affects their gene-expression
portrait [139].
REFERENCES
1. Jones PA, Martienssen R. A blueprint for a Human Epigenome
Project: the AACR Human Epigenome Workshop. Cancer Res.
200; 6:11241–11246.
2. Weber M, Davies JJ, Wittig D, et al. Chromosome-wide and pro-
moter-specific analyses identify sites of differential DNA methyla-
tion in normal and transformed human cells. Nat Genet.
2005;37:853–862.
3. Zhang X, Yazaki J, Sundaresan A, et al. Genome-wide high-reso-
lution mapping and functional analysis of DNA methylation in
arabidopsis. Cell. 2006;126:1189–1201.
4. Jacinto FV, Ballestar E, Esteller M. Methyl-DNA immunoprecipi-
tation (MeDIP): Hunting down the DNA methylome. Biotechni-
ques. 2008;44:35, 37, 39 passim.
5. Kurdistani SK, Tavazoie S, Grunstein M. Mapping global histone
acetylation patterns to gene expression. Cell. 2004;117:721–733.
6. Bernstein BE, Kamal M, Lindblad-Toh K, et al. Genomic maps
and comparative analysis of histone modifications in human
and mouse. Cell. 2005;120:169–181.
7. Azuara V, Perry P, Sauer S, et al. Chromatin signatures of plurip-
otent cell lines. Nat Cell Biol. 2006;8:532–538.
8. Esteller M. The necessity of a human epigenome project. Carci-
nogenesis. 2006;27:1121–1125.
9. Surani MA, Barton SC, Norris ML. Development of reconstituted
mouse eggs suggests imprinting of the genome during gameto-
genesis. Nature. 1984;308:548–550.
10. DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of
the mouse insulin-like growth factor II gene. Cell. 1991;64:849–859.
11. Morison IM, Ramsay JP, Spencer HG. A census of mammalian
imprinting. Trends Genet. 2005;21:457–465.
12. Dolinoy DC, Weidman JR, Jirtle RL. Epigenetic gene regulation:
Linking early developmental environment to adult disease.
Reprod Toxicol. 2007;23:297–307.
13. Murrell A, Ito Y, Verde G, et al. Distinct methylation changes at
the IGF2-H19 locus in congenital growth disorders and cancer.
PLoS ONE. 2008;3:e1849.
 Chapter 8 The Human Epigenome: Implications for the Understanding of Human Disease
14. Feil R, Berger F. Convergent evolution of genomic imprinting in
plants and mammals. Trends Genet. 2007;23:192–199.
15. Ubeda F, Wilkins JF. Imprinted genes and human disease: An
evolutionary perspective. Adv Exp Med Biol. 2008;626:101–115.
16. Kou YC, Shao L, Peng HH, et al. A recurrent intragenic genomic
duplication, other novel mutations in NLRP7 and imprinting
defects in recurrent biparental hydatidiform moles. Mol Hum
Reprod. 2008;14:33–40.
17. Oosterhuis JW, Looijenga LH. Testicular germ-cell tumours in a
broader perspective. Nat Rev Cancer. 2005;5:210–222.
18. Morison IM, Reeve AE. A catalogue of imprinted genes and par-
ent-of-origin effects in humans and animals. Hum Mol Genet.
1998;7:1599–1609.
19. Lefebvre L, Viville S, Barton SC, et al. Abnormal maternal beha-
viour and growth retardation associated with loss of the imprinted
gene Mest. Nat Genet. 1998;20:163–169.
20. Falls JG, Pulford DJ, Wylie AA, et al. Genomic imprinting: Impli-
cations for human disease. Am J Pathol. 1999;154:635–647.
21. Chen C, Visootsak J, Dills S, et al. Prader-Willi syndrome: An
update and review for the primary pediatrician. Clin Pediatr
(Phila). 2007;46:580–591.
22. Camprubi C, Coll MD, Villatoro S, et al. Imprinting center anal-
ysis in Prader-Willi and Angelman syndrome patients with typical
and atypical phenotypes. Eur J Med Genet. 2007;50:11–20.
23. Kantor B, Shemer R, Razin A. The Prader-Willi/Angelman
imprinted domain and its control center. Cytogenet Genome Res.
2006;113:300–305.
24. Maina EN, Webb T, Soni S, et al. Analysis of candidate imprinted
genes in PWS subjects with atypical genetics: A possible inactivat-
ing mutation in the SNURF/SNRPN minimal promoter. J Hum
Genet. 2007;52:297–307.
25. Watrin F, Le Meur E, Roeckel N, et al. The Prader-Willi syn-
drome murine imprinting center is not involved in the spatio-
temporal transcriptional regulation of the Necdin gene. BMC
Genet. 2005;6:1.
26. Yang T, Adamson TE, Resnick JL, et al. A mouse model for
Prader-Willi syndrome imprinting-centre mutations. Nat Genet.
1998;19:25–31.
27. Kishino T, Lalande M, Wagstaff J. UBE3A/E6-AP mutations
cause Angelman syndrome. Nat Genet. 1997;15:70–73.
28. Lalande M, Calciano MA. Molecular epigenetics of Angelman
syndrome. Cell Mol Life Sci. 2007;64:947–960.
29. Fang P, Lev-Lehman E, Tsai TF, et al. The spectrum of mutations
in UBE3A causing Angelman syndrome. Hum Mol Genet.
1999;8:129–135.
30. Meguro M, Kashiwagi A, Mitsuya K, et al. A novel maternally
expressed gene, ATP10C, encodes a putative aminophospholi-
pid translocase associated with Angelman syndrome. Nat Genet.
2001;28:19–20.
31. Herzing LB, Kim SJ, Cook EH Jr, et al. The human aminopho-
spholipid-transporting ATPase gene ATP10C maps adjacent to
UBE3A and exhibits similar imprinted expression. Am J Hum
Genet. 2001;68:1501–1505.
32. Weksberg R, Shuman C, Smith AC. Beckwith-Wiedemann syn-
drome. Am J Med Genet C Semin Med Genet. 2005;137C:12–23.
33. Scott RH, Stiller CA, Walker L, et al. Syndromes and constitu-
tional chromosomal abnormalities associated with Wilms
tumour. J Med Genet. 2006;43:705–715.
34. Cohen Jr MM. Beckwith-Wiedemann syndrome: Historical, clini-
copathological, and etiopathogenetic perspectives. Pediatr Dev
Pathol. 2005;8:287–304.
35. Cooper WN, Luharia A, Evans GA, et al. Molecular subtypes and
phenotypic expression of Beckwith-Wiedemann syndrome. Eur J
Hum Genet. 2005;13:1025–1032.
36. Joyce JA, Lam WK, Catchpoole DJ, et al. Imprinting of IGF2 and
H19: Lack of reciprocity in sporadic Beckwith-Wiedemann syn-
drome. Hum Mol Genet. 1997;6:1543–1548.
37. Higashimoto K, Soejima H, Saito T, et al. Imprinting disruption
of the CDKN1C/KCNQ1OT1 domain: The molecular mechan-
isms causing Beckwith-Wiedemann syndrome and cancer. Cyto-
genet Genome Res. 2006;113:306–312.
38. DeBaun MR, Niemitz EL, McNeil DE, et al. Epigenetic altera-
tions of H19 and LIT1 distinguish patients with Beckwith-Wiede-
mann syndrome with cancer and birth defects. Am J Hum Genet.
2002;70:604–611.
39. Cerrato F, Sparago A, Verde G, et al. Different mechanisms
cause imprinting defects at the IGF2/H19 locus in Beckwith-
Wiedemann syndrome and Wilms’ tumour. Hum Mol Genet.
2008;17:1427–1435.
40. Diaz-Meyer N, Yang Y, Sait SN, et al. Alternative mechanisms
associated with silencing of CDKN1C in Beckwith-Wiedemann
syndrome. J Med Genet. 2005;42:648–655.
41. Enklaar T, Zabel BU, Prawitt D. Beckwith-Wiedemann syndrome:
Multiple molecular mechanisms. Expert Rev Mol Med. 2006;8:1–19.
42. Feinberg AP. Phenotypic plasticity and the epigenetics of human
disease. Nature. 2007;447:433–440.
43. Baujat G, Rio M, Rossignol S, et al. Paradoxical NSD1 mutations
in Beckwith-Wiedemann syndrome and 11p15 anomalies in
Sotos syndrome. Am J Hum Genet. 2004;74:715–720.
44. Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes
of some human cancers from their normal counterparts. Nature.
1983;301:89–92.
45. Esteller M. Cancer epigenomics: DNA methylomes and histone-
modification maps. Nat Rev Genet. 2007;8:286–298.
46. Fraga MF, Ballestar E, Villar-Garea A, et al. Loss of acetylation at
Lys16 and trimethylation at Lys20 of histone H4 is a common
hallmark of human cancer. Nat Genet. 2005;37:391–400.
47. Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability
and tumors promoted by DNA hypomethylation. Science. 2003;
300:455.
48. Karpf AR, Matsui S. Genetic disruption of cytosine DNA methyl-
transferase enzymes induces chromosomal instability in human
cancer cells. Cancer Res. 2005;65:8635–8639.
49. Schulz WA. L1 retrotransposons in human cancers. J Biomed Bio-
technol. 2006;2006:83672.
50. Bjornsson HT, Brown LJ, Fallin MD, et al. Epigenetic specificity
of loss of imprinting of the IGF2 gene in Wilms tumors. J Natl
Cancer Inst. 2007;99:1270–1273.
51. Li Y, Meng G, Guo QN. Changes in genomic imprinting and
gene expression associated with transformation in a model of
human osteosarcoma. Exp Mol Pathol. 2008;84:234–239.
52. Wu J, Qin Y, Li B, et al. Hypomethylated and hypermethylated
profiles of H19DMR are associated with the aberrant imprinting
of IGF2 and H19 in human hepatocellular carcinoma. Genomics.
2008; 91:443–450.
53. Byun HM, Wong HL, Birnstein EA, et al. Examination of IGF2
and H19 loss of imprinting in bladder cancer. Cancer Res.
2007;67:10753–10758.
54. Fan T, Schmidtmann A, Xi S, et al. DNA hypomethylation
caused by Lsh deletion promotes erythroleukemia development.
Epigenetics. 2008;3:134–142.
55. Wu H, Chen Y, Liang J, et al. Hypomethylation-linked activation
of PAX2 mediates tamoxifen-stimulated endometrial carcino-
genesis. Nature. 2005;438:981–987.
56. Brueckner B, Stresemann C, Kuner R, et al. The human let-7a-3
locus contains an epigenetically regulated microRNA gene with
oncogenic function. Cancer Res. 2007;67:1419–1423.
57. Sakai T, Toguchida J, Ohtani N, et al. Allele-specific hypermethy-
lation of the retinoblastoma tumor-suppressor gene. Am J Hum
Genet. 1991;48:880–888.
58. Herman JG, Baylin SB. Gene silencing in cancer in association
with promoter hypermethylation. N Engl J Med. 2003;349:
2042–2054.
59. Esteller M, Corn PG, Baylin SB, et al. A gene hypermethylation
profile of human cancer. Cancer Res. 2001;61:3225–3229.
60. Paz MF, Fraga MF, Avila S, et al. A systematic profile of DNA
methylation in human cancer cell lines. Cancer Res. 2003;63:
1114–1121.
61. Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in
hereditary human cancers mimic sporadic tumorigenesis. Hum
Mol Genet. 2001;10:3001–3007.
62. Agrelo R, Cheng WH, Setien F, et al. Epigenetic inactivation of
the premature aging Werner syndrome gene in human cancer.
Proc Natl Acad Sci USA. 2006;103:8822–8827.
63. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:
683–692.
64. Majid S, Kikuno N, Nelles J, et al. Genistein induces the
p21WAF1/CIP1 and p16INK4a tumor suppressor genes in pros-
tate cancer cells by epigenetic mechanisms involving active chro-
matin modification. Cancer Res. 2008;68:2736–2744.
161
Part II Concepts in Molecular Biology and Genetics
65. Fraga MF, Esteller M. Towards the human cancer epigenome: A
first draft of histone modifications. Cell Cycle. 2005;4:1377–1381.
66. Seligson DB, Horvath S, Shi T, et al. Global histone modification
patterns predict risk of prostate cancer recurrence. Nature.
2005;435:1262–1266.
67. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in
gene regulation. Nat Rev Genet. 2004;5:522–531.
68. Calin GA, Croce CM. MicroRNA signatures in human cancers.
Nat Rev Cancer. 2006;6:857–866.
69. Lujambio A, Esteller M. CpG island hypermethylation of tumor
suppressor microRNAs in human cancer. Cell Cycle. 2007;6:
1455–1459.
70. Saito Y, Liang G, Egger G, et al. Specific activation of microRNA-
127 with downregulation of the proto-oncogene BCL6 by
chromatin-modifying drugs in human cancer cells. Cancer Cell.
2006;9:435–443.
71. Lujambio A, Ropero S, Ballestar E, et al. Genetic unmasking of
an epigenetically silenced microRNA in human cancer cells.
Cancer Res. 2007;67:1424–1429.
72. Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev
Cancer. 2004;4:143–153.
73. Lopez-Serra L, Ballestar E, Fraga MF, et al. A profile of methyl-CpG
binding domain protein occupancy of hypermethylated promoter
CpG islands of tumor suppressor genes in human cancer. Cancer
Res. 2006;66:8342–8346.
74. Yang XJ. The diverse superfamily of lysine acetyltransferases and
their roles in leukemia and other diseases. Nucleic Acids Res.
2004;32:959–976.
75. Intini D, Fabris S, Storlazzi T, et al. Identification of a novel IGH-
MMSET fusion transcript in a human myeloma cell line with the
t(4;14)(p16.3;q32) chromosomal translocation. Br J Haematol.
2004;126:437–439.
76. Jaju RJ, Fidler C, Haas OA, et al. A novel gene, NSD1, is fused to
NUP98 in the t(5;11)(q35;p15.5) in de novo childhood acute
myeloid leukemia. Blood. 2001;98:1264–1267.
77. Rosati R, La Starza R, Veronese A, et al. NUP98 is fused to the
NSD3 gene in acute myeloid leukemia associated with t(8;11)
(p11.2;p15). Blood. 2002;99:3857–3860.
78. Bracken AP, Pasini D, Capra M, et al. EZH2 is downstream of the
pRB-E2F pathway, essential for proliferation and amplified in
cancer. Embo J. 2003;22:5323–5335.
79. Huntsman DG, Chin SF, Muleris M, et al. MLL2, the second
human homolog of the Drosophila trithorax gene, maps to
19q13.1 and is amplified in solid tumor cell lines. Oncogene.
1999;18:7975–7984.
80. Angrand PO, Apiou F, Stewart AF, et al. NSD3, a new SET
domain-containing gene, maps to 8p12 and is amplified in
human breast cancer cell lines. Genomics. 2001;74:79–88.
81. Cloos PA, Christensen J, Agger K, et al. The putative oncogene
GASC1 demethylates tri- and dimethylated lysine 9 on histone
H3. Nature. 2006;442:307–311.
82. Ropero S, Fraga MF, Ballestar E, et al. A truncating mutation of
HDAC2 in human cancers confers resistance to histone deacety-
lase inhibition. Nat Genet. 2006;38:566–569.
83. Moinova HR, Chen WD, Shen L, et al. HLTF gene silencing
in human colon cancer. Proc Natl Acad Sci USA. 2002;99:
4562–4567.
84. Gunduz E, Gunduz M, Ouchida M, et al. Genetic and epigenetic
alterations of BRG1 promote oral cancer development. Int J
Oncol. 2005;26:201–210.
85. Ballestar E, Esteller M, Richardson BC. The epigenetic face
of systemic lupus erythematosus. J Immunol. 2006;176:7143–7147.
86. Corvetta A, Della Bitta R, Luchetti MM, et al. 5-Methylcytosine
content of DNA in blood, synovial mononuclear cells and syno-
vial tissue from patients affected by autoimmune rheumatic dis-
eases. J Chromatogr. 1991;566:481–491.
87. Kominato Y, Hata Y, Takizawa H, et al. Expression of human
histo-blood group ABO genes is dependent upon DNA methyla-
tion of the promoter region. J Biol Chem. 1999;274:37240–37250.
88. Menendez L, Walker LD, Matyunina LV, et al. Epigenetic changes
within the promoter region of the HLA-G gene in ovarian tumors.
Mol Cancer. 2008;7:43.
89. Honda T, Tamura G, Waki T, et al. Demethylation of MAGE pro-
moters during gastric cancer progression. Br J Cancer.
2004;90:838–843.
162
90. Nagarajan RP, Hogart AR, Gwye Y, et al. Reduced MeCP2
expression is frequent in autism frontal cortex and correlates
with aberrant MECP2 promoter methylation. Epigenetics. 2006;
1:e1–e11.
91. Lund G, Andersson L, Lauria M, et al. DNA methylation poly-
morphisms precede any histological sign of atherosclerosis
in mice lacking apolipoprotein E. J Biol Chem. 2004;279:
29147–29154.
92. Abel T, Zukin RS. Epigenetic targets of HDAC inhibition in
neurodegenerative and psychiatric disorders. Curr Opin Pharma-
col. 2008;8:57–64.
93. Hu N, Qiu X, Luo Y, et al. Abnormal histone modification
patterns in lupus CD4þ T cells. J Rheumatol. 2008;35:804–810.
94. Gibbons R. Alpha thalassaemia-mental retardation, X linked.
Orphanet J Rare Dis. 2006;1:15.
95. De La Fuente R, Viveiros MM, Wigglesworth K, et al. ATRX, a
member of the SNF2 family of helicase/ATPases, is required
for chromosome alignment and meiotic spindle organization
in metaphase II stage mouse oocytes. Dev Biol. 2004;272:1–14.
96. Sanlaville D, Verloes A. CHARGE syndrome: An update. Eur J
Hum Genet. 2007;15:389–399.
97. Jongmans MC, Hoefsloot LH, van der Donk KP, et al. Familial
CHARGE syndrome and the CHD7 gene: A recurrent missense
mutation, intrafamilial recurrence and variability. Am J Med
Genet A. 2008;146A:43–50.
98. Spivak G. The many faces of Cockayne syndrome. Proc Natl Acad
Sci USA. 2004;101:15273–15274.
99. Fousteri M, Vermeulen W, van Zeeland AA, et al. Cockayne syn-
drome A and B proteins differentially regulate recruitment of
chromatin remodeling and repair factors to stalled RNA poly-
merase II in vivo. Mol Cell. 2006;23:471–482.
100. Boerkoel CF, O’Neill S, Andre JL, et al. Manifestations and
treatment of Schimke immuno-osseous dysplasia: 14 new cases
and a review of the literature. Eur J Pediatr. 2000;159:1–7.
101. Boerkoel CF, Takashima H, John J, et al. Mutant chromatin
remodeling protein SMARCAL1 causes Schimke immuno-osse-
ous dysplasia. Nat Genet. 2002;30:215–220.
102. Brown DC, Grace E, Sumner AT, et al. ICF syndrome (immuno-
deficiency, centromeric instability and facial anomalies): Inves-
tigation of heterochromatin abnormalities and review of
clinical outcome. Hum Genet. 1995;96: 411–416.
103. Ehrlich M. The ICF syndrome, a DNA methyltransferase 3B
deficiency and immunodeficiency disease. Clin Immunol.
2003;109:17–28.
104. Xu GL, Bestor TH, Bourc’his D, et al. Chromosome instability
and immunodeficiency syndrome caused by mutations in a
DNA methyltransferase gene. Nature. 1999;402:187–191.
105. Moog U, Smeets EE, van Roozendaal KE, et al. Neurodevelop-
mental disorders in males related to the gene causing Rett syn-
drome in females (MECP2). Eur J Paediatr Neurol. 2003;7:5–12.
106. Van den Veyver IB, Zoghbi HY. Methyl-CpG-binding protein
2 mutations in Rett syndrome. Curr Opin Genet Dev. 2000;
10:275–279.
107. Laccone F, Junemann I, Whatley S, et al. Large deletions of the
MECP2 gene detected by gene dosage analysis in patients with
Rett syndrome. Hum Mutat. 2004;23:234–244.
108. Ballestar E, Ropero S, Alaminos M, et al. The impact of MECP2
mutations in the expression patterns of Rett syndrome patients.
Hum Genet. 2005;116:91–104.
109. Li MR, Pan H, Bao XH, et al. MECP2 and CDKL5 gene muta-
tion analysis in Chinese patients with Rett syndrome. J Hum
Genet. 2007;52:38–47.
110. Jones PL, Veenstra GJ, Wade PA, et al. Methylated DNA and
MeCP2 recruit histone deacetylase to repress transcription.
Nat Genet. 1998;19:187–191.
111. Esteller M. Rett syndrome: The first forty years: 1966–2006.
Epigenetics. 2007;2:1.
112. Abuhatzira L, Makedonski K, Kaufman Y, et al. MeCP2 defi-
ciency in the brain decreases BDNF levels by REST/CoREST-
mediated repression and increases TRKB production. Epige-
netics. 2007;2:214–222.
113. Kesler SR, Simensen RJ, Voeller K, et al. Altered neurodeve-
lopment associated with mutations of RSK2: A morphometric
MRI study of Coffin-Lowry syndrome. Neurogenetics. 2007;
8:143–147.
 Chapter 8 The Human Epigenome: Implications for the Understanding of Human Disease
114. Field M, Tarpey P, Boyle J, et al. Mutations in the RSK2
(RPS6KA3) gene cause Coffin-Lowry syndrome and nonsyn-
dromic X-linked mental retardation. Clin Genet. 2006;70:
509–515.
115. Kang S, Dong S, Guo A, et al. Epidermal growth factor stimu-
lates RSK2 activation through activation of the MEK/ERK path-
way and src-dependent tyrosine phosphorylation of RSK2 at
Tyr-529. J Biol Chem. 2008;283:4652–4657.
116. Merienne K, Jacquot S, Trivier E, et al. Rapid immunoblot and
kinase assay tests for a syndromal form of X linked mental
retardation: Coffin-Lowry syndrome. J Med Genet. 1998;35:
890–894.
117. Roelfsema JH, Peters DJ. Rubinstein-Taybi syndrome: Clinical
and molecular overview. Expert Rev Mol Med. 2007;9:1–16.
118. Bartsch O, Schmidt S, Richter M, et al. DNA sequencing of
CREBBP demonstrates mutations in 56% of patients with
Rubinstein-Taybi syndrome (RSTS) and in another patient with
incomplete RSTS. Hum Genet. 2005;117:485–493.
119. Zimmermann N, Acosta AM, Kohlhase J, et al. Confirmation of
EP300 gene mutations as a rare cause of Rubinstein-Taybi syn-
drome. Eur J Hum Genet. 2007;15:837–842.
120. Roelfsema JH, White SJ, Ariyurek Y, et al. Genetic heterogeneity
in Rubinstein-Taybi syndrome: Mutations in both the CBP and
EP300 genes cause disease. Am J Hum Genet. 2005;76:572–580.
121. Tatton-Brown K, Rahman N. Sotos syndrome. Eur J Hum Genet.
2007;15:264–271.
122. Rahman N. Mechanisms predisposing to childhood overgrowth
and cancer. Curr Opin Genet Dev. 2005;15:227–233.
123. Martinez-Glez V, Lapunzina P. Sotos syndrome is associated with
leukemia/lymphoma. Am J Med Genet A. 2007;143A:1244–1245.
124. Jaenisch R, Bird A. Epigenetic regulation of gene expression:
How the genome integrates intrinsic and environmental sig-
nals. Nat Genet. 2003;33(Suppl):245–254.
125. Waterland RA, Lin JR, Smith CA, et al. Post-weaning diet affects
genomic imprinting at the insulin-like growth factor 2 (IGF2)
locus. Hum Mol Genet. 2006;15:705–716.
126. Kirke PN, Mills JL, Molloy AM, et al. Impact of the MTHFR
C677T polymorphism on risk of neural tube defects: Case-
control study. BMJ. 2004;328:1535–1536.
127. Perez AB, D’Almeida V, Vergani N, et al. Methylenetetrahydro-
folate reductase (MTHFR): Incidence of mutations C677T and
A1298C in Brazilian population and its correlation with plasma
homocysteine levels in spina bifida. Am J Med Genet A.
2003;119A:20–25.
128. Giovannucci E. Alcohol, one-carbon metabolism, and colorec-
tal cancer: Recent insights from molecular studies. J Nutr.
2004;134:2475S–2481S.
129. Labrune P, Zittoun J, Duvaltier I, et al. Haemolytic uraemic syn-
drome and pulmonary hypertension in a patient with methio-
nine synthase deficiency. Eur J Pediatr. 1999;158:734–739.
130. Benbrahim-Tallaa L, Waterland RA, Styblo M, et al. Molecular
events associated with arsenic-induced malignant transforma-
tion of human prostatic epithelial cells: Aberrant genomic
DNA methylation and K-ras oncogene activation. Toxicol Appl
Pharmacol. 2005;206: 288–298.
131. Poirier LA, Vlasova TI. The prospective role of abnormal
methyl metabolism in cadmium toxicity. Environ Health Perspect.
2002;110(Suppl 5):793–795.
132. Silbergeld EK, Waalkes M, Rice JM. Lead as a carcinogen:
Experimental evidence and mechanisms of action. Am J Ind
Med. 2000;38:316–323.
133. Salnikow K, Costa M. Epigenetic mechanisms of nickel carcino-
genesis. J Environ Pathol Toxicol Oncol. 2000;19:307–318.
134. Wei YD, Tepperman K, Huang MY, et al. Chromium inhibits
transcription from polycyclic aromatic hydrocarbon-inducible
promoters by blocking the release of histone deacetylase and
preventing the binding of p300 to chromatin. J Biol Chem.
2004;279:4110–4119.
135. Anway MD, Cupp AS, Uzumcu M, et al. Epigenetic transgenera-
tional actions of endocrine disruptors and male fertility. Science.
2005;308:1466–1469.
136. Weaver IC, Cervoni N, Champagne FA, et al. Epigenetic pro-
gramming by maternal behavior. Nat Neurosci. 2004;7:847–854.
137. Niemitz EL, Feinberg AP. Epigenetics and assisted reproductive
technology: A call for investigation. Am J Hum Genet. 2004;74:
599–609.
138. Rideout WM, 3rd, Eggan K, Jaenisch R. Nuclear cloning and
epigenetic reprogramming of the genome. Science. 2001;293:
1093–1098.
139. Fraga MF, Ballestar E, Paz MF, et al. Epigenetic differences arise
during the lifetime of monozygotic twins. Proc Natl Acad Sci
USA. 2005;102:10604–10609.
163