**Genomic Imprinting and Human Reproduction**

I.N. Lebedev

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57241

## **1. Introduction**

The high frequency of reproductive losses is specific to the human race. The chances of conceiving and giving birth to a healthy child for women between 20 and 30 years old are estimated to be as low as 21-28% per cycle [1]. About 60% of zygotes are eliminated during the pre-implantation or early post-implantation developmental stages and 15-20% of clinically recognized pregnancies are lost during the first trimester [2]. Approximately 50-60% of spontaneously aborted embryos have chromosomal abnormalities which are not compatible with prenatal development [3]. At the same time, the death of another considerable number of embryos with normal karyotypes cannot be explained by existing cytogenetic theories. Considering ontogenesis as a result of the unrolling of the strict developmental program, the epigenetic basis of this process may be of outstanding significance.

According to the classical definition, coined by Conrad Waddington in 1942, epigenetics is a branch of biology which studies "causal mechanisms" by which "the genes of the genotype bring about phenotypic effects" [4]. In its beginning, epigenetics was a synonym for develop‐ mental genetics. However, in contrast to classical genetic theories, the subject of epigenetics has a wider diversity of phenomena, which may be unrelated to changes in gene nucleotide sequences [5]. A strong surge of interest in studying the epigenetic basis of human hereditary pathology has been noted over the last several years [6, 7]. New classes of epigenetic diseases, namely chromatin diseases [8] and imprinting disorders [9] have been identified. However, little is known about the features and nature of epigenetic abnormalities, i.e., epimutations, [10] during human prenatal development. In this chapter information on the impact of genomic imprinting abnormalities on embryo development is summarized and discussed.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Genomic imprinting and its role in embryogenesis**

Genomic imprinting is an epigenetic phenomenon, which is related to differential parent-oforigin gene expression. The term "imprinting" was taken from physiology. It was Konrad Lorenz, an Austrian zoologist, ethologist and ornithologist, who, when working with geese, rediscovered the principle of imprinting (originally described by Douglas Spalding in the 19th century) in the behaviour of nidifugous birds when a young bird acquired several of its behavioural characteristics from one parent.

triploid diploidization mechanism [16], but both haploid genomes are paternal in their nature. This bipaternal karyotype is not compatible with the development of an embryo body, but leads to a hyper proliferation of trophoblasts cells and a complete hydatidiform mole (CHM) with an increased risk of chorioepithelioma. The observed effect may be explained by the double increase in the dose of imprinted genes expressed from paternal chromosomes, which promotes proliferative and invasive activity of the trophoblasts cells as well as an absence of activity of the maternal imprinted genes, which, in turn, must suppress trophoblast prolifer‐

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The parental differences in imprinted genes expression are epigenetic in their nature. They are established during gamete differentiation by sex-dependent epigenetic chromatin modifica‐ tions, mainly by the differential DNA methylation of promoter regions of imprinted genes or regulatory imprinted centres, which are further stably inherited in the somatic cells of the progeny. These regular and consecutive alterations of chromatin organization are referred to as epigenetic genome reprogramming [17, 18] (Figure 1). This starts in the primordial germ cells when they enter the gonads. Both imprinted and non-imprinted loci become demethy‐ lated. This total erasure of epigenetic information is required for the totipontency of future germ cells, imprinting switching and for the prevention of the inheritance of epigenetic defects. The demethylated chromatin's state remains until the duration of the mitotic arrest in male germ cells and the meiotic arrest in female ones. When mitotic divisions of spermogonia are resumed, *de novo* DNA methylation begins, which terminates completely by the time of pachytene of meiosis I. In oocytes, *de novo* DNA methylation starts only at their maturation and ends by metaphase II. In this period, sex-specific methylation of imprinted genes is established. In one imprinting genes, DNA methylation occurs exclusively in oogenesis,

The second wave of epigenetic genome reprogramming, which involves somatic cells, begins immediately after fertilization. The paternal chromosomes became decondensed, protamines in the chromatin are replaced by histones and fast demethylation of paternal genome is triggered. The maternal genome undergoes slow passive demethylation. It is believed that demethylation of parental genomes is required to induce pluripotency in embryonic stem cells. Later, during implantation, *de novo* DNA methylation is launched again, which results in specific methylation patterns of particular genomic regions in different cells and tissues. This process, in fact, provides one of the most important mechanisms for committing and regulating tissue specific gene expression during ontogeny. It is significant that imprinted genes avoid this reprogramming wave in somatic cells, preserving their differential methylation pattern inherited from the parents. Genomic imprinting is also regulated through other epigenetic mechanisms, such as histone modifications, antisense transcripts and small non-coding RNA,

At present, (August 2013), it is reported that there are about 90 imprinted genes in a human genome [21]. Most of them are involved in the regulation of intrauterine foetal development through the control of cell proliferation and the differentiation of placental tissues, regulation of metabolism of some hormones and growth factors [22]. The evolutionary reverse to the haploid expression of a subset of genes in mammalian and flowering plants genomes was a

which have been discussed in detail in some comprehensive reviews [19, 20].

whereas in others it occurs during spermatogenesis [19].

ation.

The term "chromosomal imprinting" was coined in 1960 by Helen Crouse, one of only three PhD students trained by Nobel Laureate Barbara McClintock. Crouse described the selective elimination of paternal chromosomes in the male meiosis in *Sciara* fly [11]. In the first meiotic division of spermatogenesis, a monopolar spindle forms upon which all the maternally derived homologues move to a single pole and all the paternally derived homologues move away from the single pole and are completely eliminated from the cell as a nucleoplasmic bud. Crouse wrote, "the 'imprint' a chromosome bears is unrelated to the genetic construction of the chromosome and is determined only by the sex of the germ line through which the chromo‐ some has been inherited" (cited in [12]). At the time that Crouse first used the term, chromo‐ some imprinting was known to occur in *Sciara* spermatogenesis. This mechanism of selected chromosome segregation remains enigmatic, although much headway has been made in mammalian systems [12].

The first evidence of the parental genome's memory in mammals came from experiments conducted by the Surani, McGrath and Solter groups with pronuclei transplantation in mouse zygotes in 1984 [13, 14]. These studies were aimed at answering the question about the absence of parthenogenesis in mammalian reproduction. It was discovered that diploid androgenic mouse embryos derived from zygotes, which contained two paternal pronuclei and none of the maternal pronuclei, demonstrated an extensive proliferation of extraembryonic tissues but poor development of the embryo *per se*, which usually did not reach a 4-6 somite stage. In contrast, gynogenic zygotes with two maternal pronuclei and an absence of the paternal genome resulted in embryos which developed until the stage of early somites, but then died due to the poor development of supportive extraembryonic tissues. These pioneers' studies established that diploidy alone is not sufficient for embryonic development, but that a balance of maternal and paternal genomes is strongly required for normal embryogenesis. Moreover, the impact of parental genomes on embryo development is different. It seems that the maternal genome is responsible for the development of the embryo body to a greater extent, whereas the paternal one is involved in the support of extraembryonic tissue differentiation.

It is interesting to note that a similar effect of the increasing number of paternal genomes in the zygote is also observed in humans due to an abnormality of fertilization [15]. Fertilization of a diploid oocyte by normal haploid sperm or double fertilization of a normal oocyte by two haploid sperms leads to triploidy in the zygote. In this case, the partial hydatidiform mole (PHM) arises. PHM is characterized by the cystic degeneration of chorionic villi and the presence of a visible embryo in the foetal sac. In the case of the extrusion of the maternal pronucleus from such a triploid zygote, a diploid karyotype is restored through the postzygotic triploid diploidization mechanism [16], but both haploid genomes are paternal in their nature. This bipaternal karyotype is not compatible with the development of an embryo body, but leads to a hyper proliferation of trophoblasts cells and a complete hydatidiform mole (CHM) with an increased risk of chorioepithelioma. The observed effect may be explained by the double increase in the dose of imprinted genes expressed from paternal chromosomes, which promotes proliferative and invasive activity of the trophoblasts cells as well as an absence of activity of the maternal imprinted genes, which, in turn, must suppress trophoblast prolifer‐ ation.

**2. Genomic imprinting and its role in embryogenesis**

behavioural characteristics from one parent.

mammalian systems [12].

122 Epigenetics and Epigenomics

Genomic imprinting is an epigenetic phenomenon, which is related to differential parent-oforigin gene expression. The term "imprinting" was taken from physiology. It was Konrad Lorenz, an Austrian zoologist, ethologist and ornithologist, who, when working with geese, rediscovered the principle of imprinting (originally described by Douglas Spalding in the 19th century) in the behaviour of nidifugous birds when a young bird acquired several of its

The term "chromosomal imprinting" was coined in 1960 by Helen Crouse, one of only three PhD students trained by Nobel Laureate Barbara McClintock. Crouse described the selective elimination of paternal chromosomes in the male meiosis in *Sciara* fly [11]. In the first meiotic division of spermatogenesis, a monopolar spindle forms upon which all the maternally derived homologues move to a single pole and all the paternally derived homologues move away from the single pole and are completely eliminated from the cell as a nucleoplasmic bud. Crouse wrote, "the 'imprint' a chromosome bears is unrelated to the genetic construction of the chromosome and is determined only by the sex of the germ line through which the chromo‐ some has been inherited" (cited in [12]). At the time that Crouse first used the term, chromo‐ some imprinting was known to occur in *Sciara* spermatogenesis. This mechanism of selected chromosome segregation remains enigmatic, although much headway has been made in

The first evidence of the parental genome's memory in mammals came from experiments conducted by the Surani, McGrath and Solter groups with pronuclei transplantation in mouse zygotes in 1984 [13, 14]. These studies were aimed at answering the question about the absence of parthenogenesis in mammalian reproduction. It was discovered that diploid androgenic mouse embryos derived from zygotes, which contained two paternal pronuclei and none of the maternal pronuclei, demonstrated an extensive proliferation of extraembryonic tissues but poor development of the embryo *per se*, which usually did not reach a 4-6 somite stage. In contrast, gynogenic zygotes with two maternal pronuclei and an absence of the paternal genome resulted in embryos which developed until the stage of early somites, but then died due to the poor development of supportive extraembryonic tissues. These pioneers' studies established that diploidy alone is not sufficient for embryonic development, but that a balance of maternal and paternal genomes is strongly required for normal embryogenesis. Moreover, the impact of parental genomes on embryo development is different. It seems that the maternal genome is responsible for the development of the embryo body to a greater extent, whereas

the paternal one is involved in the support of extraembryonic tissue differentiation.

It is interesting to note that a similar effect of the increasing number of paternal genomes in the zygote is also observed in humans due to an abnormality of fertilization [15]. Fertilization of a diploid oocyte by normal haploid sperm or double fertilization of a normal oocyte by two haploid sperms leads to triploidy in the zygote. In this case, the partial hydatidiform mole (PHM) arises. PHM is characterized by the cystic degeneration of chorionic villi and the presence of a visible embryo in the foetal sac. In the case of the extrusion of the maternal pronucleus from such a triploid zygote, a diploid karyotype is restored through the postzygotic

The parental differences in imprinted genes expression are epigenetic in their nature. They are established during gamete differentiation by sex-dependent epigenetic chromatin modifica‐ tions, mainly by the differential DNA methylation of promoter regions of imprinted genes or regulatory imprinted centres, which are further stably inherited in the somatic cells of the progeny. These regular and consecutive alterations of chromatin organization are referred to as epigenetic genome reprogramming [17, 18] (Figure 1). This starts in the primordial germ cells when they enter the gonads. Both imprinted and non-imprinted loci become demethy‐ lated. This total erasure of epigenetic information is required for the totipontency of future germ cells, imprinting switching and for the prevention of the inheritance of epigenetic defects. The demethylated chromatin's state remains until the duration of the mitotic arrest in male germ cells and the meiotic arrest in female ones. When mitotic divisions of spermogonia are resumed, *de novo* DNA methylation begins, which terminates completely by the time of pachytene of meiosis I. In oocytes, *de novo* DNA methylation starts only at their maturation and ends by metaphase II. In this period, sex-specific methylation of imprinted genes is established. In one imprinting genes, DNA methylation occurs exclusively in oogenesis, whereas in others it occurs during spermatogenesis [19].

The second wave of epigenetic genome reprogramming, which involves somatic cells, begins immediately after fertilization. The paternal chromosomes became decondensed, protamines in the chromatin are replaced by histones and fast demethylation of paternal genome is triggered. The maternal genome undergoes slow passive demethylation. It is believed that demethylation of parental genomes is required to induce pluripotency in embryonic stem cells. Later, during implantation, *de novo* DNA methylation is launched again, which results in specific methylation patterns of particular genomic regions in different cells and tissues. This process, in fact, provides one of the most important mechanisms for committing and regulating tissue specific gene expression during ontogeny. It is significant that imprinted genes avoid this reprogramming wave in somatic cells, preserving their differential methylation pattern inherited from the parents. Genomic imprinting is also regulated through other epigenetic mechanisms, such as histone modifications, antisense transcripts and small non-coding RNA, which have been discussed in detail in some comprehensive reviews [19, 20].

At present, (August 2013), it is reported that there are about 90 imprinted genes in a human genome [21]. Most of them are involved in the regulation of intrauterine foetal development through the control of cell proliferation and the differentiation of placental tissues, regulation of metabolism of some hormones and growth factors [22]. The evolutionary reverse to the haploid expression of a subset of genes in mammalian and flowering plants genomes was a

of imprinted genes localized on these chromosomal regions. Moreover, spontaneous abortions without previous cytogenetic analysis were included in some studies that could have led to overestimation the obtained rate. As a result, the frequency of UPD for chromosomes, which contain known imprinted genes in spontaneous abortions were estimated to be 1% (3/305) or 1.14 per 1,000 occasions of chromosome inheritance from parents to progeny. The latter figure was obtained from the investigation of 6,156 cases of chromosome inheritance by DNA microsatellite analyses and seven cases of UPD were found in the eight cited studies. This figure does not significantly differ from the expected frequency (1.65:1,000), predicted from data about frequencies of chromosome segregation errors in gametogenesis and early em‐

Thus, it seems that UPD is a selectively neutral phenomenon in human reproduction. More‐ over, UPD for some chromosomes (6, 7, 11, 14 and 15) are compatible with postnatal life leading

18 0 [26] - first trimester, without cytogenetic



Total: 305 7 (2,3%)

**UPD cases References**

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[25]

[28]

[29]

[30]

UPD(21)mat, UPD(21)mat in combination with trisomies 7 and 9

> UPD(9)mat UPD(21)mat

Maternal segmental heterosomy 16pter-D16S3107 and isodisomy D16S3018-qter

> Segmental UPD(14q)pat, UPD(7q)mat

52\* 0 [31]\*

bryogenesis, which can lead to uniparental inheritance in humans [33].

**Number of spontaneous abortions**

23

**Samples**

Anembryonic pregnancies without




**Table 1.** Results of UPD studies in spontaneous abortions

Missed abortions and anembryonic


**Note:** \* - samples partially overlap.

pregnancies:

cytogenetic analysis

analysis

Spontaneous abortions:

**Figure 1.** Dynamics of epigenetic genome reprogramming

great surprise. Several hypotheses were introduced to explain this intriguing fact, but the "sex conflict" was one the most popular among them [23]. According to this hypothesis, maternal imprinted genes in mammals are responsible for the suppression of foetal growth in order to save maternal resources for subsequent pregnancies. In contrast, paternal imprinted genes are involved in the promotion of foetal growth that provides higher chances of survival for many offspring.

Testing this hypothesis in mouse models, the direct evidence for the significant role of genomic imprinting in mammalian embryo development was obtained. The generation of uniparental disomies (UPD) in progeny of translocation carrier's mice gives nonviable embryos [24]. This fact leads to the idea of searching for UPD in human spontaneous abortions in order to estimate the impact of genomic imprinting abnormalities on prenatal death.

## **3. UPD in spontaneous abortions**

To date, eight studies have been performed to find UPD in spontaneous abortions [25-32] (Table 1). However, the obtained results were modest. Only seven cases of UPD (2.3%) among a total of 305 spontaneous abortions were found and most of them involved chromosomes which did not contain known imprinted genes. Only in three cases segmental UPD (16p/16q (mat), 14q (pat) and 7q (mat)) embryo death can be connected with a disturbance of the dose of imprinted genes localized on these chromosomal regions. Moreover, spontaneous abortions without previous cytogenetic analysis were included in some studies that could have led to overestimation the obtained rate. As a result, the frequency of UPD for chromosomes, which contain known imprinted genes in spontaneous abortions were estimated to be 1% (3/305) or 1.14 per 1,000 occasions of chromosome inheritance from parents to progeny. The latter figure was obtained from the investigation of 6,156 cases of chromosome inheritance by DNA microsatellite analyses and seven cases of UPD were found in the eight cited studies. This figure does not significantly differ from the expected frequency (1.65:1,000), predicted from data about frequencies of chromosome segregation errors in gametogenesis and early em‐ bryogenesis, which can lead to uniparental inheritance in humans [33].

Thus, it seems that UPD is a selectively neutral phenomenon in human reproduction. More‐ over, UPD for some chromosomes (6, 7, 11, 14 and 15) are compatible with postnatal life leading


**Table 1.** Results of UPD studies in spontaneous abortions

great surprise. Several hypotheses were introduced to explain this intriguing fact, but the "sex conflict" was one the most popular among them [23]. According to this hypothesis, maternal imprinted genes in mammals are responsible for the suppression of foetal growth in order to save maternal resources for subsequent pregnancies. In contrast, paternal imprinted genes are involved in the promotion of foetal growth that provides higher chances of survival for many


**CP #1 CP #2 CP #3 CP #4 CP #5** 

**Fertilization Cleavage Implantation** 


**Embryo** 

**Trophectoderm** 

Testing this hypothesis in mouse models, the direct evidence for the significant role of genomic imprinting in mammalian embryo development was obtained. The generation of uniparental disomies (UPD) in progeny of translocation carrier's mice gives nonviable embryos [24]. This fact leads to the idea of searching for UPD in human spontaneous abortions in order to estimate

To date, eight studies have been performed to find UPD in spontaneous abortions [25-32] (Table 1). However, the obtained results were modest. Only seven cases of UPD (2.3%) among a total of 305 spontaneous abortions were found and most of them involved chromosomes which did not contain known imprinted genes. Only in three cases segmental UPD (16p/16q (mat), 14q (pat) and 7q (mat)) embryo death can be connected with a disturbance of the dose

the impact of genomic imprinting abnormalities on prenatal death.


**Mitosis G0 Meiosis** 


**Figure 1.** Dynamics of epigenetic genome reprogramming

**Differentiation** 

**3. UPD in spontaneous abortions**

offspring.

**Level of CpG methylation** 

**Primordial germ cells** 

124 Epigenetics and Epigenomics

**Migration** 

CP – critical periods of reprogramming

to a formation of specific genomic imprinting disorders: transient neonatal diabetes mellitus (TNDM), Silver-Russell syndrome (SRS), Beckwith-Wiedemann syndrome (BWS), Wang and Temple syndromes, Prader-Willi syndrome (PWS) and Angelman syndrome (AS), respective‐ ly [34]. It became clear that UPD is a rare cytogenetic phenomenon, which cannot explain the mechanisms of imprinted genes disturbances in human pregnancy loss. The only evidence for the pathogenetic role of genomic imprinting abnormalities in human reproduction remains from studies on a hydatidiform mole which originated from the doubling of paternal genome in conception [15]. However, this conclusion does not offer an answer on the possible mech‐ anisms of imprinting disturbances associated with early pregnancy loss.

On the other hand, the change of the imprinted gene dose may be achieved by epimutations, i.e., abnormal methylation of the expressed allele or demethylation of the silenced allele. From a functional point of view, epimutations and UPD influences on imprinted genes expression should be similar (Figure 2). It is important that epimutation on a single allele is enough to

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Before the testing of the hypothesis about the influence of methylation defects in imprinted genes on the aetiology of early pregnancy loss, a classification of epimutations was introduced [36]. They were divided into the following several groups depending on their germinal or somatic origin, hyper- or hypomethylation of active or silenced alleles, and affected parental

1.1. Epimutations causing a global disturbance of genomic imprinting at the genome level.

1.2. Epimutations at the imprinting centres causing a disturbance of imprinting of neighbouring genes.

2.1.1. Errors of genomic imprinting erasure in primordial germ cells with retention of methyl groups (Critical Period 1, CP #1, on Figure 1). These errors may lead to transgenerational inheritance

2.1.2.1. Absence of methylation of imprinted genes alleles that normally should be

2.1.2.2. Aberrant methylation of imprinted genes alleles that normally should be

2.2.1. Abnormal hypomethylation of inactive parental alleles of imprinted genes during epigenetic

2.2.2. Abnormal methylation of expressed parental alleles of imprinted genes during de novo DNA

2.2.3. Spontaneous hypomethylation of inactive parental alleles of imprinted genes in somatic cells

2.2.4. Spontaneous hypermethylation of expressed parental alleles of imprinted genes in somatic

2.1.2. Errors of imprinting establishment during gametogenesis (CP #2 on Figure 1)

methylation upon epigenetic genome reprogramming (CP #4 on Figure 1).

after epigenetic genome reprogramming (CP #5 on Figure 1).

3.1. Hypomethylation of the inactive maternal allele of the imprinted gene. 3.2. Hypomethylation of the inactive paternal allele of the imprinted gene. 3.3. Hypermethylation of the expressed maternal allele of the imprinted gene. 3.4. Hypermethylation of the expressed paternal allele of the imprinted gene.

cells after epigenetic genome reprogramming (CP #5 on Figure 1). 3. Types of epimutations of imprinted genes by their functional consequences and affected parental alleles:

achieve the imprinted gene dysfunction in a dominant manner.

1. Types of epimutations of imprinted genes by the loci involved:

1.3. Epimutations at the imprinted genes. 2. Types of epimutations of imprinted genes by their origin:

2.1. Germinal epimutations

2.2. Somatic epimutations

of epigenetic defects.

methylated in sperm or oocytes.

unmethylated in sperm or oocytes.

genome reprogramming (CP #3 on Figure 1).

chromosomes (Table 2):

## **4. Epimutations of imprinted genes in spontaneous abortions**

Taking into account the epigenetic nature of genomic imprinting, we proposed a hypothesis that expected the deleterious effect of abnormal imprinted genes expression to be visible at the epigenetic rather than cytogenetic level [35]. Indeed, UPD formation requires a combination of several subsequent errors in chromosomal segregation during parental meiosis, fertilization and embryo development. For example, the most frequent mechanism of UPD formation is trisomy rescue. It arises from chromosomal nondisjunction in meiosis, trisomy formation in the zygote after fertilization and the loss of additional chromosome in some somatic cells during subsequent mitotic divisions. In a third of the cases of such correction, the situation of inheritance of both homologues from one parent may be observed. If involved chromosome contains an imprinted gene, then the double increase or complete loss of expression of imprinted genes may be detected and it is dependent on the parental origin of expressed allele.

**Figure 2.** Effects of UPD and epimutations on expression of imprinted genes.

On the other hand, the change of the imprinted gene dose may be achieved by epimutations, i.e., abnormal methylation of the expressed allele or demethylation of the silenced allele. From a functional point of view, epimutations and UPD influences on imprinted genes expression should be similar (Figure 2). It is important that epimutation on a single allele is enough to achieve the imprinted gene dysfunction in a dominant manner.

Before the testing of the hypothesis about the influence of methylation defects in imprinted genes on the aetiology of early pregnancy loss, a classification of epimutations was introduced [36]. They were divided into the following several groups depending on their germinal or somatic origin, hyper- or hypomethylation of active or silenced alleles, and affected parental chromosomes (Table 2):

	- 1.1. Epimutations causing a global disturbance of genomic imprinting at the genome level.
	- 1.2. Epimutations at the imprinting centres causing a disturbance of imprinting of neighbouring genes.
	- 1.3. Epimutations at the imprinted genes.
	- 2.1. Germinal epimutations

to a formation of specific genomic imprinting disorders: transient neonatal diabetes mellitus (TNDM), Silver-Russell syndrome (SRS), Beckwith-Wiedemann syndrome (BWS), Wang and Temple syndromes, Prader-Willi syndrome (PWS) and Angelman syndrome (AS), respective‐ ly [34]. It became clear that UPD is a rare cytogenetic phenomenon, which cannot explain the mechanisms of imprinted genes disturbances in human pregnancy loss. The only evidence for the pathogenetic role of genomic imprinting abnormalities in human reproduction remains from studies on a hydatidiform mole which originated from the doubling of paternal genome in conception [15]. However, this conclusion does not offer an answer on the possible mech‐

Taking into account the epigenetic nature of genomic imprinting, we proposed a hypothesis that expected the deleterious effect of abnormal imprinted genes expression to be visible at the epigenetic rather than cytogenetic level [35]. Indeed, UPD formation requires a combination of several subsequent errors in chromosomal segregation during parental meiosis, fertilization and embryo development. For example, the most frequent mechanism of UPD formation is trisomy rescue. It arises from chromosomal nondisjunction in meiosis, trisomy formation in the zygote after fertilization and the loss of additional chromosome in some somatic cells during subsequent mitotic divisions. In a third of the cases of such correction, the situation of inheritance of both homologues from one parent may be observed. If involved chromosome contains an imprinted gene, then the double increase or complete loss of expression of imprinted genes may be detected and it is dependent on the parental origin of expressed allele.

**UPD pat** 

**CH3 CH3 CH3 CH3**

**Epimutation (hypomethylation of imprinted gene on maternal chromosome)** 

anisms of imprinting disturbances associated with early pregnancy loss.

**Normal cells** 

**Figure 2.** Effects of UPD and epimutations on expression of imprinted genes.

**CH3**

126 Epigenetics and Epigenomics

**4. Epimutations of imprinted genes in spontaneous abortions**

2.1.1. Errors of genomic imprinting erasure in primordial germ cells with retention of methyl groups (Critical Period 1, CP #1, on Figure 1). These errors may lead to transgenerational inheritance of epigenetic defects.

2.1.2. Errors of imprinting establishment during gametogenesis (CP #2 on Figure 1)

2.1.2.1. Absence of methylation of imprinted genes alleles that normally should be methylated in sperm or oocytes.

2.1.2.2. Aberrant methylation of imprinted genes alleles that normally should be unmethylated in sperm or oocytes.

2.2. Somatic epimutations

2.2.1. Abnormal hypomethylation of inactive parental alleles of imprinted genes during epigenetic genome reprogramming (CP #3 on Figure 1).

2.2.2. Abnormal methylation of expressed parental alleles of imprinted genes during de novo DNA methylation upon epigenetic genome reprogramming (CP #4 on Figure 1).

2.2.3. Spontaneous hypomethylation of inactive parental alleles of imprinted genes in somatic cells after epigenetic genome reprogramming (CP #5 on Figure 1).

2.2.4. Spontaneous hypermethylation of expressed parental alleles of imprinted genes in somatic cells after epigenetic genome reprogramming (CP #5 on Figure 1).

	- 3.1. Hypomethylation of the inactive maternal allele of the imprinted gene.
	- 3.2. Hypomethylation of the inactive paternal allele of the imprinted gene.
	- 3.3. Hypermethylation of the expressed maternal allele of the imprinted gene.
	- 3.4. Hypermethylation of the expressed paternal allele of the imprinted gene.


complete mole. Subsequent studies revealed that BiCHM arose due to germinal epimutations, namely the absence of *de novo* methylation of imprinted genes in oogenesis (type # 2.1.2.1 according to classification) [38]. However, these findings do not offer an answer about the prevalence of imprinting defects in human reproductive losses. The convenient model system

**CH3 CH3 CH3 CH3**

**CH3 CH3 CH3 CH3**

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for studies became spontaneous abortions.

**Epigenotypes of parents (P1, P2)** 

**Erasing of parental epigenotypes in gametes** 

**Establishment of imprinting in gametes** 

**CH3**

**CH3**

**Epigenotypes of progeny (F1)** 

> **Normal cells**

**Figure 3.** Cytogenetic and epigenetic mechanisms of hydatidiform mole formation.

differential methylation of parental alleles of this imprinted gene.

**Androgenic complete hydatidiform mole** 

The first study devoted to the analysis of methylation status of *SNRPN* imprinted gene was published by our group in 2006 [39]. However, it was shown that all investigated spontaneous abortions, as well as the control sample of induced (therapeutic) abortions, had normal

Our further studies on imprinted genes *PLAGL1 (ZAC), CDKN1C* and regulatory imprinting centres *IGF2/H19* and *KCNQ1OT1* (*LIT1*) in spontaneous abortions revealed that 9.5% and 10.3% of embryos with normal karyotype had hypomethylation of *KCNQ1OT1* and *PLAGL1*,

**CH3 CH3 CH3 CH3**

**Biparental complete hydatidiform mole** 

**CH3 CH3**

*Absence of methylation* 

**Table 2.** Types of epimutations of imprinted genes in human (according to [36] with modifications)

The first evidence of epimutations in imprinted genes in reproductive losses came from studies of biparental complete hydatidiform mole (BiCHM, MIM #231090). This pathology, as opposed to classical androgenic complete mole, arose in the case of normal biparental karyotype [37] (Figure 3). It was shown that imprinted genes, which are methylated on maternal chromo‐ somes in normal embryos, became hypomethylated in the case of BiCHM both on the paternal and maternal homologues [15]. Functionally, this epigenetic status is the same as an androgenic complete mole. Subsequent studies revealed that BiCHM arose due to germinal epimutations, namely the absence of *de novo* methylation of imprinted genes in oogenesis (type # 2.1.2.1 according to classification) [38]. However, these findings do not offer an answer about the prevalence of imprinting defects in human reproductive losses. The convenient model system for studies became spontaneous abortions.

**Type of epimutations**

128 Epigenetics and Epigenomics

(CP #1)

(CP #2)

Abnormal hypermethylation of expressed parental alleles of imprinted genes during epigenetic genome reprogramming (CP #4)

Errors of genomic imprinting erasure with retention of methyl groups

Absence of imprinted genes alleles methylation (CP #2)

Aberrant hypermethylation of imprinted genes alleles

Abnormal hypomethylation of inactive parental alleles of imprinted genes during epigenetic genome reprogramming (CP #3)

Stochastic epimutations

(hypo- and hypermethylation) in somatic cells after epigenetic genome reprogramming (CP #5)

Germinal epimutations

Somatic epimutations

**Abnormal hypomethylation of Abnormal hypermethylation of maternal allele paternal allele maternal allele paternal allele**

> *IGF2/H19* (BWS)

> *IGF2/H19* (BWS)

> *IGF2/H19* (BWS)

*IGF2/H19* (Wilm's tumour);

*P73* (acute leukaemia, Burkitt's lymphoma);

*DLK1/GTL2* (renal carcinoma)

*SNURF-SNRPN* (PWS);

*PEG1/MEST* (SRS)

*SNURF-SNRPN* (PWS); *PEG1/MEST*

*SNURF-SNRPN* (PWS)

*SNURF-SNRPN* (mosaic forms of PWS);

*PEG3* (cervical and endometrial

*PLAGL1* (ovarian cancer)

(SRS)

Not applicable Not applicable

Not applicable Not applicable

Not applicable Not applicable

Not applicable Not applicable

Not applicable Not applicable

**Table 2.** Types of epimutations of imprinted genes in human (according to [36] with modifications)

*IGF2/H19* (SRS)

Partial hypomethylation of *IGF2/H19* (SRS)

*P73* (renal carcinoma; lung cancer)

The first evidence of epimutations in imprinted genes in reproductive losses came from studies of biparental complete hydatidiform mole (BiCHM, MIM #231090). This pathology, as opposed to classical androgenic complete mole, arose in the case of normal biparental karyotype [37] (Figure 3). It was shown that imprinted genes, which are methylated on maternal chromo‐ somes in normal embryos, became hypomethylated in the case of BiCHM both on the paternal and maternal homologues [15]. Functionally, this epigenetic status is the same as an androgenic

*PEG3* carcinomas); (choriocarcinoma)

BiCHM;

*SNURF-SNRPN* (AS); *KCNQ1OT1* (BWS, TNDM); *PLAGL1* (TNDM, BWS)

*SNURF-SNRPN* (mosaic forms of AS).

mosaic forms of TNDM

*KCNQ1OT1* (oesophagus carcinoma, liver cancer);

MHS;

**Figure 3.** Cytogenetic and epigenetic mechanisms of hydatidiform mole formation.

The first study devoted to the analysis of methylation status of *SNRPN* imprinted gene was published by our group in 2006 [39]. However, it was shown that all investigated spontaneous abortions, as well as the control sample of induced (therapeutic) abortions, had normal differential methylation of parental alleles of this imprinted gene.

Our further studies on imprinted genes *PLAGL1 (ZAC), CDKN1C* and regulatory imprinting centres *IGF2/H19* and *KCNQ1OT1* (*LIT1*) in spontaneous abortions revealed that 9.5% and 10.3% of embryos with normal karyotype had hypomethylation of *KCNQ1OT1* and *PLAGL1*, respectively, on maternal chromosomes [35, 40]. It is interesting that in two embryos from the investigated group (2.3%) epimutations were detected in both genes. In both families, the women had not had a successfully delivered pregnancy. In one family, the woman had had four spontaneous abortions, in the other, the woman had had two spontaneous abortions and a stillbirth. It was shown that the frequency of recurrent pregnancy loss (i.e., the loss of three or more consecutive pregnancies) was significantly higher in woman who had had a loss of methylation in *PLAGL1* in spontaneous abortions tissues, in comparison with woman without epimutations in the aborted embryo (33% and 8%, respectively, p < 0.05). A similar tendency was observed for *KCNQ1OT1* epimutations, but it does not reach a statistically significant difference.

*ZNF331,* and *CDKAL1* was decreased. In this study, hypomethylation of *DLK1, H19,* and *SNRPN* was demonstrated also in the placental tissues of foetuses with IUGR. In another study, expression levels of four imprinted genes (*IGF2, PEG10, PHLDA2,* and *CDKN1C*) were investigated in embryonic and placental tissues of 38 spontaneous abortions [49]. The in‐ creased expression of *PHLDA2* was found both in the embryonic and placental tissues in the first trimester. During the second trimester, elevated expression levels of all four genes were observed in both tissues, however, for the *PHLDA2* gene it was specific to the embryonic tissues only. A decreased expression of *PEG3* was detected in embryonic tissues during the third

trimester.

Sample size Tissue

SA 55 МТ

SB 57 МТ

SA 87 CC

SA 13 CC

SA 13 EM

SA 165 CC

SA 29 CC

406

Total

MLMD

frequency

3.6%

17.5%

2.3% (2)

100% (13)

100% (13)

3.6%

10.3%

12.1% (49)

5.0% (5/100)

46.2% (12/26)

34.6% (9/26)

MLMD is indicated in parentheses); ↓ – hypomethylation; ↑ – hypermethylation.

7.1% (11/154)

3.2% (13/406)

**Table 3.** Frequency of epimutations in paternally expressed imprinted genes in spontaneous abortions

34.6% (9/26)

**Notes for tables 3 and 4:** SA – spontaneous abortion; SB – stillbirth; МТ – muscular tissues of embryo; CC – chorion cytotrophoblast; EM – extraembryonic mesoderm; MLMD – multilocus methylation defects (number of embryos with

7.4% (30/406)

*PLAGL1* (6q24)

*PEG10*

(7q21)

*DLK1* (14q32)

*PEG3* (19q13.4)

*LIT1* (11p15)

*INS* (11p15)

*SNRPN* (15q11)

*WT1* (11p15)

(2) - - - 2↑ 1↑ - 1↑ - - - - - - - [47]

(10) - - - 6↑ 2↑ - 9↑ - - - - - - - [47]

2↓ - - - 2↓ - 0 - - - - - - - [40]

1↓ 3↓ 0 0 0 2↑ 2↑ 4↓ 5↓ 3↓ 2↓ 1↑ 0 3↑ [50]

2↓ 9↓ 9↓ 0 0 7↑ 10↑ 10↓ 3↓ 5↓ 6↓ 5↑ 0 7↑ [50]

(6) - - - - 6↑ - 7↑ - - - - - - - [46]

(3) - - - 1↓, 2↑ 2↑ - 1↑ - - - - - 1↑ - [51]

53.8% (14/26)

↓ - 16.0% (65/406); ↑ - 18.7% (76/406)

30.8% (8/26)

30.8% (8/26)

30.8% (8/26)

23.1% (6/26)

1.8% (1/55)

38.5% (10/26)

*KCNQ1* (11p15)

*GABRB3* (15q11)

*HTR2A* (13q14)

*TRPM5* (11p15)

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*MEST*

(7q32)

*GABRA5*

(15q11)

Ref.

131

Pathology

It was remarkable also that all detected epimutations were confined to one placental tissue (cytotrophoblast or extraembryonic mesoderm) only indicating their somatic origin in postimplantation stages of development after the divergence of embryonic and extraembryonic cell lineages. This observation has an important value for the discussion on the increase of genomic imprinting disorders in children born after the application of assisted reproductive technologies (ART) [20, 41-44]. Indeed, several cases of children with BWS, SRS and AS born after *in vitro* fertilization (IVF) or intracytoplasmic sperm injection (ICSI) have been reported. However, it is more significant that all patients with confirmed molecular diagnosis had hypomethylation of imprinted genes on maternal chromosomes as a cause of disorder among all the other known possible mechanisms which can alter imprinted genes expression – UPD, chromosomal microdeletions or microduplications, point mutations in imprinted genes or regulatory imprinted centres. This fact is supported by results of *in vitro* studies in model organisms [45]. This often leads to a hasty one-sided conclusion that manipulations with gametes and embryos in an artificial medium cannot support correct recording and mainte‐ nance of genomic imprinting and they are responsible for an increase of imprinting defects in children after ART. Our, and other, recently published data from spontaneous abortions studies [46] indicates that the same epimutations with relatively high frequency can be observed in embryos from natural cycles also and not only from IVF or ICSI pregnancies. This means that epimutations of imprinted genes arising during human embryo development, and infertile pairs or couples with the history of recurrent pregnancy losses (typical patients of ART clinics), may have increasing chances to produce progeny with epimutations after overcoming the natural reproductive barriers by IVF or ICSI. This conclusion implies that the nature of epimutations involves the interaction of hereditary and environmental factors.

Evidence for epimutations of imprinted genes in spontaneous abortions was also noted in other subsequent studies (See tables 3 and 4). For example, multiple hypermethylation of imprinted genes was detected in 4% (2 out of 55) of spontaneous abortions and 18% (10 out of 57) of stillbirths [47]. In this study, *H19, KCNQ1OT1, PEG3* and *SNRPN* genes with paternal expres‐ sion and *MEG3* and *NESP55* with maternal expression were investigated. In other studies, the expression level of several imprinted genes was compared between placental tissues from normal pregnancies and pregnancies complicated by intrauterine growth retardation (IUGR) [48]. It was found that *PHLDA2, ILK2, NNAT, CCDC86* and *PEG10* genes had increased expression in IUGR pregnancies, whereas the expression level of *PLAGL1, DHCR24,* *ZNF331,* and *CDKAL1* was decreased. In this study, hypomethylation of *DLK1, H19,* and *SNRPN* was demonstrated also in the placental tissues of foetuses with IUGR. In another study, expression levels of four imprinted genes (*IGF2, PEG10, PHLDA2,* and *CDKN1C*) were investigated in embryonic and placental tissues of 38 spontaneous abortions [49]. The in‐ creased expression of *PHLDA2* was found both in the embryonic and placental tissues in the first trimester. During the second trimester, elevated expression levels of all four genes were observed in both tissues, however, for the *PHLDA2* gene it was specific to the embryonic tissues only. A decreased expression of *PEG3* was detected in embryonic tissues during the third trimester.

respectively, on maternal chromosomes [35, 40]. It is interesting that in two embryos from the investigated group (2.3%) epimutations were detected in both genes. In both families, the women had not had a successfully delivered pregnancy. In one family, the woman had had four spontaneous abortions, in the other, the woman had had two spontaneous abortions and a stillbirth. It was shown that the frequency of recurrent pregnancy loss (i.e., the loss of three or more consecutive pregnancies) was significantly higher in woman who had had a loss of methylation in *PLAGL1* in spontaneous abortions tissues, in comparison with woman without epimutations in the aborted embryo (33% and 8%, respectively, p < 0.05). A similar tendency was observed for *KCNQ1OT1* epimutations, but it does not reach a statistically significant

It was remarkable also that all detected epimutations were confined to one placental tissue (cytotrophoblast or extraembryonic mesoderm) only indicating their somatic origin in postimplantation stages of development after the divergence of embryonic and extraembryonic cell lineages. This observation has an important value for the discussion on the increase of genomic imprinting disorders in children born after the application of assisted reproductive technologies (ART) [20, 41-44]. Indeed, several cases of children with BWS, SRS and AS born after *in vitro* fertilization (IVF) or intracytoplasmic sperm injection (ICSI) have been reported. However, it is more significant that all patients with confirmed molecular diagnosis had hypomethylation of imprinted genes on maternal chromosomes as a cause of disorder among all the other known possible mechanisms which can alter imprinted genes expression – UPD, chromosomal microdeletions or microduplications, point mutations in imprinted genes or regulatory imprinted centres. This fact is supported by results of *in vitro* studies in model organisms [45]. This often leads to a hasty one-sided conclusion that manipulations with gametes and embryos in an artificial medium cannot support correct recording and mainte‐ nance of genomic imprinting and they are responsible for an increase of imprinting defects in children after ART. Our, and other, recently published data from spontaneous abortions studies [46] indicates that the same epimutations with relatively high frequency can be observed in embryos from natural cycles also and not only from IVF or ICSI pregnancies. This means that epimutations of imprinted genes arising during human embryo development, and infertile pairs or couples with the history of recurrent pregnancy losses (typical patients of ART clinics), may have increasing chances to produce progeny with epimutations after overcoming the natural reproductive barriers by IVF or ICSI. This conclusion implies that the nature of epimutations involves the interaction of hereditary and environmental factors.

Evidence for epimutations of imprinted genes in spontaneous abortions was also noted in other subsequent studies (See tables 3 and 4). For example, multiple hypermethylation of imprinted genes was detected in 4% (2 out of 55) of spontaneous abortions and 18% (10 out of 57) of stillbirths [47]. In this study, *H19, KCNQ1OT1, PEG3* and *SNRPN* genes with paternal expres‐ sion and *MEG3* and *NESP55* with maternal expression were investigated. In other studies, the expression level of several imprinted genes was compared between placental tissues from normal pregnancies and pregnancies complicated by intrauterine growth retardation (IUGR) [48]. It was found that *PHLDA2, ILK2, NNAT, CCDC86* and *PEG10* genes had increased expression in IUGR pregnancies, whereas the expression level of *PLAGL1, DHCR24,*

difference.

130 Epigenetics and Epigenomics


**Notes for tables 3 and 4:** SA – spontaneous abortion; SB – stillbirth; МТ – muscular tissues of embryo; CC – chorion cytotrophoblast; EM – extraembryonic mesoderm; MLMD – multilocus methylation defects (number of embryos with MLMD is indicated in parentheses); ↓ – hypomethylation; ↑ – hypermethylation.

**Table 3.** Frequency of epimutations in paternally expressed imprinted genes in spontaneous abortions


*H19* and on maternal alleles of *INS*, *TRPM5*, *PWCR1*, *GABRA5* genes. The majority of epimu‐ tations (78%) were confined to single placental tissue (extraembryonic mesoderm or cytotro‐

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133

Summarizing the published data, it is possible to note that MLMD were observed in 49 of 406 investigated spontaneous abortions (12%) (See tables 3 and 4). It is evident that this value, of a significant magnitude greater than UPD frequency in miscarriages, indicates an appreciable effect of epigenetic defects on imprinted genes in pregnancy losses. The incidence of epimu‐ tations for different genes varied from 1.8% (*MEST*) to 53.8% (*WT1*). For genes expressed from maternal chromosomes, the frequency of epimutations was 17.7%, with 10.3 % of these presented by the hypomethylation of respective inactive paternal alleles and 7.4% by the hypermethylation of active maternal alleles. For genes expressed from the paternal allele, the total frequency of epimutations was twice as frequent (34.7%). These epimutations were presented by the hypomethylation of respective inactive maternal alleles (16%) and the

The results of the studies suggest possible mechanisms of the selective influence of epimuta‐ tions of imprinted genes on early embryo development. As it was mentioned early, the "sex conflict" hypothesis is the most popular in explaining the imprinted mode of gene expression. From its point of view, the expected suppressive effect of epimutations should emerge from the hypomethylation of paternal alleles, which leads to a loss of imprinting and the biallelic expression of maternal genes responsible for foetal growth suppression, as well as from hypermethylation of paternal alleles, which leads to the absence of products responsible for foetal growth stimulation. The total incidence of such types of epimutations in spontaneous abortions is 29% (10.3 + 18.7). At the same time, the total incidence of epimutations, which can lead to the promotion of foetal growth (hypo- and hypermethylation of maternal alleles), was 23.4% (16 + 17.4). The differences between frequencies of "suppressive" and "promotion" epimutations were not statistically significant (p = 0.07). However, this is a relative estimation for several reasons, including complete acceptance of the "sex conflict" hypothesis for each imprinted gene (maternal genes are suppressors, paternal genes are activators) and the idea that there is a strong reverse correlation between gene methylation and its expression. To obtain more precise and weighted estimations, further studies of imprinted gene functions and

Does MLMD arise spontaneously in different loci or is it driven by mutations in a candidate's genes responsible for genomic imprinting establishment and maintenance? To answer this question the evidence of MLMD in patients with imprinting disorders should be discussed.

**5. Multiple methylation defects in patients with imprinting disorders**

The first evidence of multiple epimutations of imprinted genes was obtained in 2005 in the comparative analysis of methylation status of two genes – *ZAC (PLAGL1)* and *LIT1 (KCNQ1OT1)* in eight patients with BWS and 17 patients with TNDM [54]. The idea for this study was based on the fact that there is partial overlapping of some clinical features in these

phoblast), indicating postzygotic errors in imprinting maintenance in somatic cells.

hypermethylation of expressed paternal alleles (18.7%).

molecular mechanisms of their expression regulation are encouraged.

**Table 4.** Frequency of epimutations in maternally expressed imprinted genes in spontaneous abortions

It is important to note that all the above mentioned studies were performed by the "candidate gene" approach, i.e., only several interesting genes were tested based on their supposed functions in embryogenesis. A new era in this area of research began with the application of array technologies. They provide comprehensive and unbiased analysis of the human imprintome. Results of the first studies revealed a new, intriguing phenomenon of multilocus methylation defects (MLMD) on imprinted genes. This effect is presented by multiple epimu‐ tations (hypo- and/or hypermethylation) affecting several imprinted genes simultaneously in the genome in different combinations. In fact, the first example of MLMD is a BiCHM, which results from errors of imprinting establishment on maternal chromosomes during epigenetic genome reprogramming in oocytes leading to the hypomethylation of maternal alleles of different imprinted genes [38]. MLMD were detected in human reproductive losses [50] as well as in patients with genomic imprinting disorders [52]. Importantly, in one recent study 15 (8%) probands among 194 patients with clinical features of an imprinting disorder but no molecular diagnosis had methylation anomalies, including missed and unexpected molecular diagnosis [53].

Evidence of MLMD in 13 first trimester spontaneous abortions were obtained in our study by DNA methylation array the GoldenGate Methylation Cancer Panel I (Illumina) analysis of 51 imprinted genes [50]. Multiple methylation defects affecting from four to 12 genes in each embryo were found. Epimutations were presented by the hypomethylation of paternal alleles of *GRB10*, *CPA4*, *PHLDA2*, *ZNF215* genes, and maternal alleles of *PEG10*, *PLAGL1*, *WT1*, *HTR2A*, *DLK1*, *GABRB3*, *KCNQ1* genes. Hypermethylation was observed on paternal allele of *H19* and on maternal alleles of *INS*, *TRPM5*, *PWCR1*, *GABRA5* genes. The majority of epimu‐ tations (78%) were confined to single placental tissue (extraembryonic mesoderm or cytotro‐ phoblast), indicating postzygotic errors in imprinting maintenance in somatic cells.

**Pathology Sample**

132 Epigenetics and Epigenomics

Total 406

diagnosis [53].

**size**

SB 57 МТ

**Tissue**

**MLMD frequenc y**

17.5%

12.1% (49)

23.1% (6/26)

50.0% (13/26)

**Table 4.** Frequency of epimutations in maternally expressed imprinted genes in spontaneous abortions

It is important to note that all the above mentioned studies were performed by the "candidate gene" approach, i.e., only several interesting genes were tested based on their supposed functions in embryogenesis. A new era in this area of research began with the application of array technologies. They provide comprehensive and unbiased analysis of the human imprintome. Results of the first studies revealed a new, intriguing phenomenon of multilocus methylation defects (MLMD) on imprinted genes. This effect is presented by multiple epimu‐ tations (hypo- and/or hypermethylation) affecting several imprinted genes simultaneously in the genome in different combinations. In fact, the first example of MLMD is a BiCHM, which results from errors of imprinting establishment on maternal chromosomes during epigenetic genome reprogramming in oocytes leading to the hypomethylation of maternal alleles of different imprinted genes [38]. MLMD were detected in human reproductive losses [50] as well as in patients with genomic imprinting disorders [52]. Importantly, in one recent study 15 (8%) probands among 194 patients with clinical features of an imprinting disorder but no molecular diagnosis had methylation anomalies, including missed and unexpected molecular

Evidence of MLMD in 13 first trimester spontaneous abortions were obtained in our study by DNA methylation array the GoldenGate Methylation Cancer Panel I (Illumina) analysis of 51 imprinted genes [50]. Multiple methylation defects affecting from four to 12 genes in each embryo were found. Epimutations were presented by the hypomethylation of paternal alleles of *GRB10*, *CPA4*, *PHLDA2*, *ZNF215* genes, and maternal alleles of *PEG10*, *PLAGL1*, *WT1*, *HTR2A*, *DLK1*, *GABRB3*, *KCNQ1* genes. Hypermethylation was observed on paternal allele of

42.3% (11/26)

*GRB10 (7q21)*

*CPA4 (7q32)* *PHLDA2 (11p15)*

SA 55 МТ 3.6% (2) - - - - 0 1↑ 0 [47]

SA 87 CC 2.3% (2) - - - - 0 0 - [40] SA 13 EM 100% (13) 1↓ 8↓ 2↓ 8↓ 1↑ 0 0 [50] SA 13 CC 100% (13) 5↓ 5↓ 9↓ 4↓ 6↑ 0 0 [50] SA 165 CC 3.6% (6) - - - - 4↑ - - [46] SA 29 CC 10.3% (3) - - - - 0 0 0 [51]

*ZNF215 (11p15)*

(10) - - - - 4↑ 8↑ 6↑ [47]

46.2% (12/26)

↓ - 10.3% (42/406); ↑ - 7.4% (30/406)

3.7% (15/406)

3.7% (9/241)

3.9% (6/154)

*H19 (11p15)*

*MEG3 (14q32)*

*NESP55 (20q13.3)*

**Ref.**

Summarizing the published data, it is possible to note that MLMD were observed in 49 of 406 investigated spontaneous abortions (12%) (See tables 3 and 4). It is evident that this value, of a significant magnitude greater than UPD frequency in miscarriages, indicates an appreciable effect of epigenetic defects on imprinted genes in pregnancy losses. The incidence of epimu‐ tations for different genes varied from 1.8% (*MEST*) to 53.8% (*WT1*). For genes expressed from maternal chromosomes, the frequency of epimutations was 17.7%, with 10.3 % of these presented by the hypomethylation of respective inactive paternal alleles and 7.4% by the hypermethylation of active maternal alleles. For genes expressed from the paternal allele, the total frequency of epimutations was twice as frequent (34.7%). These epimutations were presented by the hypomethylation of respective inactive maternal alleles (16%) and the hypermethylation of expressed paternal alleles (18.7%).

The results of the studies suggest possible mechanisms of the selective influence of epimuta‐ tions of imprinted genes on early embryo development. As it was mentioned early, the "sex conflict" hypothesis is the most popular in explaining the imprinted mode of gene expression. From its point of view, the expected suppressive effect of epimutations should emerge from the hypomethylation of paternal alleles, which leads to a loss of imprinting and the biallelic expression of maternal genes responsible for foetal growth suppression, as well as from hypermethylation of paternal alleles, which leads to the absence of products responsible for foetal growth stimulation. The total incidence of such types of epimutations in spontaneous abortions is 29% (10.3 + 18.7). At the same time, the total incidence of epimutations, which can lead to the promotion of foetal growth (hypo- and hypermethylation of maternal alleles), was 23.4% (16 + 17.4). The differences between frequencies of "suppressive" and "promotion" epimutations were not statistically significant (p = 0.07). However, this is a relative estimation for several reasons, including complete acceptance of the "sex conflict" hypothesis for each imprinted gene (maternal genes are suppressors, paternal genes are activators) and the idea that there is a strong reverse correlation between gene methylation and its expression. To obtain more precise and weighted estimations, further studies of imprinted gene functions and molecular mechanisms of their expression regulation are encouraged.

Does MLMD arise spontaneously in different loci or is it driven by mutations in a candidate's genes responsible for genomic imprinting establishment and maintenance? To answer this question the evidence of MLMD in patients with imprinting disorders should be discussed.

## **5. Multiple methylation defects in patients with imprinting disorders**

The first evidence of multiple epimutations of imprinted genes was obtained in 2005 in the comparative analysis of methylation status of two genes – *ZAC (PLAGL1)* and *LIT1 (KCNQ1OT1)* in eight patients with BWS and 17 patients with TNDM [54]. The idea for this study was based on the fact that there is partial overlapping of some clinical features in these syndromes. This observation may reflect possible interactions between *PLAGL1* and *P57KIP2* genes. According to the author's suggestion, an overexpression of *PLAGL1* due to the loss of methylation of inactive maternal allele may lead to the suppression or inhibition of cyclindependent kinases *P57KIP2* through the hyperactivation of *LIT1 (KCNQ1OT1)*. It was shown that all eight BWS patients had normal *PLAGL1* methylation, whereas in two patients with TNDM hypomethylation at *LIT1* was detected. The first patient had UPD(6)pat. The second one had a normal karyotype, but, typically for TNDM, a loss of methylation at *PLAGL1* on maternal chromosome 6. It is interesting that in the latter patient's situation, clinical signs of TNDM were supplemented with umbilical hernia and macroglassia that are typical for BWS.

*SNURF-SNRPN* [57]. In some patients with SRS, hypomethylation at *GRB10* and *IGF2/H19* was observed [58, 59]. Multiple epimutations were observed at *H19, PEG3*, *NESPAS* and *GNAS*

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135

It was mentioned earlier that microarray technologies allow us to obtain comprehensive data sets about the methylation status of imprinted genes over the whole genome. In a recent study, 65 patients with different genomic imprinting disorders (BWS, SRS, PWS, AS, TNDM and pseudohypoparathyroidism (PHP-1B)) were investigated by using "GoldenGate Cancer Panel I" (Illumina) DNA methylation microarray [61]. MLMD were detected in all the diseases except PWS and AS, which demonstrated methylation defects at *SNRPN* only. Multiple epimutations were observed in 33% of BWS patients with *KCNQ1OT1* hypomethylation, 75% of TNDM patients with *PLAGL1* hypomethylation, 50% of PHP-1B patients with *GNASXL/EX1A* hypomethylation, and in 17% of SRS patients with *H19* hypomethylation. MLMD involved an

Thus, examinations of patients with imprinting disorders indicate that the epigenetic basis of these diseases may, in some cases, be supplemented by multiple methylation defects in several other imprinting genes in addition to epimutation at the disease-specific gene, which is responsible for the pathogenesis of major clinical features of a given syndrome. The incidence of MLMD in different imprinting disorders varies from 8.9% (SRS) to 56.3% (TNDM) (Table 5). The incidence of methylation defects at different genes varies also. The *KCNQ1OT1, H19,* and *GNAS/NESPAS* genes are the most frequently involved in MLMD. On the other hand, *SNURF-SNRPN* imprinting centre is a very rare subject in the study of methylation defects in BWS and SRS patients, and scarcely noted in the list of MLMD affected genes in patients with

Is the combination of imprinted genes affected by MLMD in different syndromes and preg‐ nancy losses non-random? Are there any specific features of nucleotide sequences and mechanisms of expression regulation of imprinted genes with different incidence of methyl‐ ation defects? Does an interaction between imprinted genes that may lead to formation of specific epigenotype and phenotype exist? Further studies are necessary to obtain answers to these questions. However, there is some data which indicates the existence of coordination mechanisms in the regulation of epigenetic status of human imprintome. For example, it was shown that epigenetic changes in the cases of BiCHM and TNDM affected imprinted genes on maternal chromosomes only, whereas other non-imprinted genes were not a subject for epimutations [68, 69]. On the other hand, multiple methylation defects were observed by DNA methylation array analysis both in imprinted and non-imprinted genes in spontaneous abortions [50]. It is also possible that MLMD may have a different molecular nature in comparison with a methylation defect at a single locus. For example, multiple methylation defects in patients with BWS were observed only in the cases of *KCNQ1OT1* hypomethylation, but not in the ones with *H19/IGF2* hypermethylation (Table 5). The possible regulation genetic mechanisms for epigenetic status of imprinted genes are discussed in the last part of the

genes in one patient with overlapping clinical symptoms of PWS and BWS [60].

additional one to 16 imprinted genes in each patient.

TNDM and MHS.

chapter.

In 2006, Mackay and colleagues reported on a study of two patients with TNDM and IUGR who had hypomethylation of *ZAC* and *KCNQ1OT1* on maternal chromosomes 6 and 11 respectively [55]. Significantly, both patients did not have the overgrowth which is typical for BWS in the case of *KCNQ1OT1* hypomethylation. However, they had moderate macroglassia, which is not typical for TNDM, and abdomen wall defects and exomphalos that are frequent in both syndromes.

Later in 2006, Mackay and colleagues described another 12 patients with TNDM and hypo‐ methylation of *ZAC* on maternal chromosome 6q24 [56]. In six patients (50%) additional methylation defects were found in different imprinted genes. It was hypomethylation of *GRB10, PEG1/MEST, KCNQ1OT1,* and *PEG3* on maternal chromosomes in different combina‐ tions. All patients with multiple methylation defects had higher birth weight and were more phenotypically diverse than other TNDM patients with different genetic aetiologies, except for *ZAC* hypomethylation, presumably reflecting the influence of dysregulation of multiple imprinted genes. It was proposed the existence of a "maternal hypomethylation syndrome" (MHS), when a patient with the loss of methylation at one maternally-methylated locus may also manifest DNA methylation loss at other loci, potentially complicated or even confounded the clinical presentation [56].

Another specific finding of this study which is also very intriguing, is that the level of mosai‐ cism for the DNA methylation index varied in different investigated tissues (blood, mouth‐ brushes, fibroblasts) and affected different genes. The presence of mosaicism indicates postzygotyic errors of imprinting maintenance in somatic cells. However, it is more significant that such errors affected only maternally, but not paternally inherited alleles. This type of epigenetic mosaicism may explain another interesting fact that all patients with MHS had a major clinical manifestation of TNDM but no other genomic imprinting disorders that can be expected from the involvement of different imprinted genes. It is reasonable to assume that germinal epimutations at *ZAC*, which in theory must be presented (or at least maintained) in all somatic cells, should lead to TNDM symptoms, whereas somatic epimutations at other different genes can partially modify only a clinical manifestation of the "main" hereditary imprinting syndrome.

Later, MLMD were also reported in other imprinting disorders. However, both maternal and paternal alleles were affected in comparison with MHS. Hypermethylation and hypomethy‐ lation of imprinted genes were reported also. For example, in some cases of BWS, loss of methylation at *KCNQ1OT1* was accompanied by hypomethylation at *PLAGL1, PEG/MEST* and *SNURF-SNRPN* [57]. In some patients with SRS, hypomethylation at *GRB10* and *IGF2/H19* was observed [58, 59]. Multiple epimutations were observed at *H19, PEG3*, *NESPAS* and *GNAS* genes in one patient with overlapping clinical symptoms of PWS and BWS [60].

syndromes. This observation may reflect possible interactions between *PLAGL1* and *P57KIP2* genes. According to the author's suggestion, an overexpression of *PLAGL1* due to the loss of methylation of inactive maternal allele may lead to the suppression or inhibition of cyclindependent kinases *P57KIP2* through the hyperactivation of *LIT1 (KCNQ1OT1)*. It was shown that all eight BWS patients had normal *PLAGL1* methylation, whereas in two patients with TNDM hypomethylation at *LIT1* was detected. The first patient had UPD(6)pat. The second one had a normal karyotype, but, typically for TNDM, a loss of methylation at *PLAGL1* on maternal chromosome 6. It is interesting that in the latter patient's situation, clinical signs of TNDM were supplemented with umbilical hernia and macroglassia that are typical for BWS. In 2006, Mackay and colleagues reported on a study of two patients with TNDM and IUGR who had hypomethylation of *ZAC* and *KCNQ1OT1* on maternal chromosomes 6 and 11 respectively [55]. Significantly, both patients did not have the overgrowth which is typical for BWS in the case of *KCNQ1OT1* hypomethylation. However, they had moderate macroglassia, which is not typical for TNDM, and abdomen wall defects and exomphalos that are frequent

Later in 2006, Mackay and colleagues described another 12 patients with TNDM and hypo‐ methylation of *ZAC* on maternal chromosome 6q24 [56]. In six patients (50%) additional methylation defects were found in different imprinted genes. It was hypomethylation of *GRB10, PEG1/MEST, KCNQ1OT1,* and *PEG3* on maternal chromosomes in different combina‐ tions. All patients with multiple methylation defects had higher birth weight and were more phenotypically diverse than other TNDM patients with different genetic aetiologies, except for *ZAC* hypomethylation, presumably reflecting the influence of dysregulation of multiple imprinted genes. It was proposed the existence of a "maternal hypomethylation syndrome" (MHS), when a patient with the loss of methylation at one maternally-methylated locus may also manifest DNA methylation loss at other loci, potentially complicated or even confounded

Another specific finding of this study which is also very intriguing, is that the level of mosai‐ cism for the DNA methylation index varied in different investigated tissues (blood, mouth‐ brushes, fibroblasts) and affected different genes. The presence of mosaicism indicates postzygotyic errors of imprinting maintenance in somatic cells. However, it is more significant that such errors affected only maternally, but not paternally inherited alleles. This type of epigenetic mosaicism may explain another interesting fact that all patients with MHS had a major clinical manifestation of TNDM but no other genomic imprinting disorders that can be expected from the involvement of different imprinted genes. It is reasonable to assume that germinal epimutations at *ZAC*, which in theory must be presented (or at least maintained) in all somatic cells, should lead to TNDM symptoms, whereas somatic epimutations at other different genes can partially modify only a clinical manifestation of the "main" hereditary

Later, MLMD were also reported in other imprinting disorders. However, both maternal and paternal alleles were affected in comparison with MHS. Hypermethylation and hypomethy‐ lation of imprinted genes were reported also. For example, in some cases of BWS, loss of methylation at *KCNQ1OT1* was accompanied by hypomethylation at *PLAGL1, PEG/MEST* and

in both syndromes.

134 Epigenetics and Epigenomics

the clinical presentation [56].

imprinting syndrome.

It was mentioned earlier that microarray technologies allow us to obtain comprehensive data sets about the methylation status of imprinted genes over the whole genome. In a recent study, 65 patients with different genomic imprinting disorders (BWS, SRS, PWS, AS, TNDM and pseudohypoparathyroidism (PHP-1B)) were investigated by using "GoldenGate Cancer Panel I" (Illumina) DNA methylation microarray [61]. MLMD were detected in all the diseases except PWS and AS, which demonstrated methylation defects at *SNRPN* only. Multiple epimutations were observed in 33% of BWS patients with *KCNQ1OT1* hypomethylation, 75% of TNDM patients with *PLAGL1* hypomethylation, 50% of PHP-1B patients with *GNASXL/EX1A* hypomethylation, and in 17% of SRS patients with *H19* hypomethylation. MLMD involved an additional one to 16 imprinted genes in each patient.

Thus, examinations of patients with imprinting disorders indicate that the epigenetic basis of these diseases may, in some cases, be supplemented by multiple methylation defects in several other imprinting genes in addition to epimutation at the disease-specific gene, which is responsible for the pathogenesis of major clinical features of a given syndrome. The incidence of MLMD in different imprinting disorders varies from 8.9% (SRS) to 56.3% (TNDM) (Table 5). The incidence of methylation defects at different genes varies also. The *KCNQ1OT1, H19,* and *GNAS/NESPAS* genes are the most frequently involved in MLMD. On the other hand, *SNURF-SNRPN* imprinting centre is a very rare subject in the study of methylation defects in BWS and SRS patients, and scarcely noted in the list of MLMD affected genes in patients with TNDM and MHS.

Is the combination of imprinted genes affected by MLMD in different syndromes and preg‐ nancy losses non-random? Are there any specific features of nucleotide sequences and mechanisms of expression regulation of imprinted genes with different incidence of methyl‐ ation defects? Does an interaction between imprinted genes that may lead to formation of specific epigenotype and phenotype exist? Further studies are necessary to obtain answers to these questions. However, there is some data which indicates the existence of coordination mechanisms in the regulation of epigenetic status of human imprintome. For example, it was shown that epigenetic changes in the cases of BiCHM and TNDM affected imprinted genes on maternal chromosomes only, whereas other non-imprinted genes were not a subject for epimutations [68, 69]. On the other hand, multiple methylation defects were observed by DNA methylation array analysis both in imprinted and non-imprinted genes in spontaneous abortions [50]. It is also possible that MLMD may have a different molecular nature in comparison with a methylation defect at a single locus. For example, multiple methylation defects in patients with BWS were observed only in the cases of *KCNQ1OT1* hypomethylation, but not in the ones with *H19/IGF2* hypermethylation (Table 5). The possible regulation genetic mechanisms for epigenetic status of imprinted genes are discussed in the last part of the chapter.


**6. Genetic control of epigenetic status of imprinted genes**

absent in the 348 individuals from the control group.

only.

The story begins again with BiCHM. Recurrent cases of this pathology or classical CHM and PHM in anamnesis, the occurrence of several cases within one pedigree, and the appearance in consanguineous couples provide evidence for an autosomal recessive mode of inheritance of BiCHM by maternal lineage. The first candidate's genes were DNA methyltransferases, however, the sequencing of it in women with a history of BiCHM did not reveal the presence of mutations. Subsequent genome-wide association studies and homozigosity mapping indicated the linkage of BiCHM with chromosomal segment 19q13.42, in which the *NLRP7 (NALP7)* gene was mapped [70]. In this first study, five mutations of *NLRP7* in familial and recurrent cases of hydatidiform mole were reported. Two mutations, IVS3+1G-A and IVS7+1G-, were found in introns 3 and 7, respectively. Three single nucleotide changes were detected in another three families: p.R693W, p.R693P, and p.N913S. All these mutations were

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137

Homozygous c.295G>T (p.Glu99X) and heterozygous c.1970A>T (p.Asp657Val) mutations were observed in a woman with four hydatidiform moles [71]. Her sister with two moles and brother were compound heterozygotes for these mutations. Her father had a homozygous p.Asp657Val mutation. Her mother had a homozygous p.Glu99X mutation, three successful pregnancies and a stillbirth in anamnesis. In another studied family, a brother and his three sisters with recurrent hydatidiform moles (two, three, and five cases in anamnesis, respec‐ tively) were homozygous for p.Arg693Pro mutations, but the brother did not have any reproductive problems. The authors of this study made a very important conclusion that mutations of *NLRP7* do not affect the foetal development in the case of paternal inheritance,

Several studies combined methylation analysis of imprinted genes and the search for *NLRP7* mutations. For example, homozygous c.2248C>G (p.L750V), c.2471+1G>A missense mutations and heterozygous c.2248C>G (p.Leu750Val), c.2810+2T>G *NLRP7* mutations in women with BiCHM and multiple hypomethylation at the *PEG3, SNRPN, KCNQ1OT1, GNAS* imprinted genes were reported [72]. Studying 11 families with BiCHM, Hayward and colleagues performed methylation analysis of the *ZAC, GNAS-NESP55*, *LIT1/KCNQ1OT1,* and *SGCE/ PEG10* genes in four families. Hypomethylation of maternal alleles at the *ZAC* and *LIT1* as well as hypermethylation of maternal alleles at the *NESP55*, which is unusual for BiCHM, were observed in all four families. The sequencing of *NLRP7* revealed eight not previously described mutations: p.K116X, p.L398R, p.S673X, p.W778X, c.939\_952dup14, c.1456dupG, c.2030delT, and c.277+1G>C [73]. Moreover, p.L398R was associated with multiple epimutations of imprinted genes indicating that some mutations in *NLRP7* may be connected with different types of methylation defects, but not restricted by the hypomethylation of maternal alleles

A search for *NLRP7* mutations was performed in 40 Tunisian families with sporadic hydati‐ diform moles [74, 75]. Two sisters in one family had a homozygous mutation p.E570X. Heterozygous mutations were found in 11 patients. There were several new mutations among them: c.544G>A (p.Val182Met), c.1480G>A (p.Ala494Thr), c.1532A>G (p.Lys511Arg) and c.

but lead to recurrent hydatidiform moles when transmitted from the mother.

**Note:** MLMD – multilocus methylation defects (number of patients with MLMD is indicated in parentheses); n.a. – not analysed; ↓ – hypomethylation; ↑ – hyperme‐ thylation.

**Table 5.** Incidence of methylation defects in patients with imprinting disorders

## **6. Genetic control of epigenetic status of imprinted genes**

**Syndrome**

**Sample size**

136 Epigenetics and Epigenomics

TNDM 12

TNDM 4

*Total 16*

**Disease-specific**

**epimutation**

*PLAGL1* hypometh

*PLAGL1* hypometh

*PLAGL1 hypometh*

*Total 267 LIT1 hypometh 25.5% (68)*

*Total 168 H19 hypometh 8.9% (15)*

*SNRPN* hypometh

PWS/BWS 1

*Total 476*

thylation.

**MLMD frequency**

*56.3% (9) 100%*

7.9% (18/227)

2.1% (2/103)

*10.3% (38/370)*

**Table 5.** Incidence of methylation defects in patients with imprinting disorders

*20.4% (97/476)* *PLAGL1 (6q24)*

*MEST/ PEG1 (7q32)*

*31.4% (5/16)*

8.6% (23/267)

2.5% (5/168)

*7.4% (35/475)* *LIT1 (11p15)*

*18.8% (3/16)*

*100%*

4.3% (7/168)

PHP-1В 10 *GNAS* hypometh 50% (5) 0 2↓ 0 0 0 1↓ 0 0 10↓ [61]

*58.4% (278/476)*

**Note:** MLMD – multilocus methylation defects (number of patients with MLMD is indicated in parentheses); n.a. – not analysed; ↓ – hypomethylation; ↑ – hyperme‐

BWS 20 *H19* hypermeth 0 0 0 0 0 0 0 0 20↑ 0 [63] SRS 23 *H19* hypometh 8.2% (2) 0 0 1↓ 0 n.a. 1↓, 2↑ 1↓ 23↓ 2↓ [66] SRS 74 *H19* hypometh 9.5% (7) 2↓ 3↓ 3↓ 5↓ 1↓ 2↓ n.a. 74↓ n.a. [64] SRS 65 *H19* hypometh 7.7% (5) n.a. 1↓ 3↓ 1↓ n.a. 3↓ 0 65↓ n.a. [67] SRS 6 *H19* hypometh 16.6%(1) 0 1↓ 0 0 0 0 0 6↓ 0 [61]

0.9% (2/227)

3.7% (6/168)

*1.8% (8/435)*

1.2% (3/256)

1.4% (1/80)

– n.a. n.a. 1↓ n.a. 1↓ n.a. n.a. 1↓ 1↓ [60]

*1.1% (4/377)*

↓ – 75.6% (360/476); ↑ – 0.6% (3/476) ↓ – 52.9% (252/476); ↑ – 4.8% (23/476)

BWS 40 *LIT1* hypometh 25% (10) n.a. 3↓ 40↓ n.a. 1↓ 6↓ n.a. n.a. n.a. [62] BWS 81 *LIT1* hypometh 21% (17) 7↓ 6↓ 81↓ 0 0 6↓ 4↓ 0 10↓ [63] BWS 68 *LIT1* hypometh 23.5% (16) 6↓ 6↓ 68↓ 0 1↓ 10↓ n.a. 1↓ n.a. [64] BWS 11 *LIT1* hypometh 45.5% (5) 0 1↓ 11↓ 2↓ n.a. 2↓ 2↓ 2↓ 2↓ [65] BWS 24 *LIT1* hypometh 25% (6) 2↓ 4↓ 24↓ 0 1↓ n.a. n.a. n.a. n.a. [57] BWS 43 *LIT1* hypometh 33% (14) 3↓ 3↓ 43↓ 0 0 3↓ 3↓ 2↓ 3↑ [61]

*DLK1/ GTL2 (14q32)*

*SNRPN (15q11)*

**Genes with paternal expression Genes with maternal expression**

50% (6) 12↓ 5↓ 3↓ 0 0 n.a. 3↓ 0 n.a. [56]

75% (3) 4↓ 0 0 0 0 1↓,1↑ 1↓ 0 0 [61]

*0 0*

*IGF2R* **(6q25)**

*12.5% (2/16)*

*10.9% (27/243)*

4.9% (8/168)

*8.7% (38/439)* *GRB10/ MEG1 (7p13)*

*25.0% (4/16)*

6.7% (9/135)

1.1% (1/94)

*5.2% (14/269)*

2.5% (5/203)

*100%*

*47.1% (194/412)*

11.9% (16/135)

> 8.7% (2/29)

*15.0% (29/193)*

*H19 (11p15)*

*0 0*

*GNAS/ NESPAS (20q13)*

**Ref.**

The story begins again with BiCHM. Recurrent cases of this pathology or classical CHM and PHM in anamnesis, the occurrence of several cases within one pedigree, and the appearance in consanguineous couples provide evidence for an autosomal recessive mode of inheritance of BiCHM by maternal lineage. The first candidate's genes were DNA methyltransferases, however, the sequencing of it in women with a history of BiCHM did not reveal the presence of mutations. Subsequent genome-wide association studies and homozigosity mapping indicated the linkage of BiCHM with chromosomal segment 19q13.42, in which the *NLRP7 (NALP7)* gene was mapped [70]. In this first study, five mutations of *NLRP7* in familial and recurrent cases of hydatidiform mole were reported. Two mutations, IVS3+1G-A and IVS7+1G-, were found in introns 3 and 7, respectively. Three single nucleotide changes were detected in another three families: p.R693W, p.R693P, and p.N913S. All these mutations were absent in the 348 individuals from the control group.

Homozygous c.295G>T (p.Glu99X) and heterozygous c.1970A>T (p.Asp657Val) mutations were observed in a woman with four hydatidiform moles [71]. Her sister with two moles and brother were compound heterozygotes for these mutations. Her father had a homozygous p.Asp657Val mutation. Her mother had a homozygous p.Glu99X mutation, three successful pregnancies and a stillbirth in anamnesis. In another studied family, a brother and his three sisters with recurrent hydatidiform moles (two, three, and five cases in anamnesis, respec‐ tively) were homozygous for p.Arg693Pro mutations, but the brother did not have any reproductive problems. The authors of this study made a very important conclusion that mutations of *NLRP7* do not affect the foetal development in the case of paternal inheritance, but lead to recurrent hydatidiform moles when transmitted from the mother.

Several studies combined methylation analysis of imprinted genes and the search for *NLRP7* mutations. For example, homozygous c.2248C>G (p.L750V), c.2471+1G>A missense mutations and heterozygous c.2248C>G (p.Leu750Val), c.2810+2T>G *NLRP7* mutations in women with BiCHM and multiple hypomethylation at the *PEG3, SNRPN, KCNQ1OT1, GNAS* imprinted genes were reported [72]. Studying 11 families with BiCHM, Hayward and colleagues performed methylation analysis of the *ZAC, GNAS-NESP55*, *LIT1/KCNQ1OT1,* and *SGCE/ PEG10* genes in four families. Hypomethylation of maternal alleles at the *ZAC* and *LIT1* as well as hypermethylation of maternal alleles at the *NESP55*, which is unusual for BiCHM, were observed in all four families. The sequencing of *NLRP7* revealed eight not previously described mutations: p.K116X, p.L398R, p.S673X, p.W778X, c.939\_952dup14, c.1456dupG, c.2030delT, and c.277+1G>C [73]. Moreover, p.L398R was associated with multiple epimutations of imprinted genes indicating that some mutations in *NLRP7* may be connected with different types of methylation defects, but not restricted by the hypomethylation of maternal alleles only.

A search for *NLRP7* mutations was performed in 40 Tunisian families with sporadic hydati‐ diform moles [74, 75]. Two sisters in one family had a homozygous mutation p.E570X. Heterozygous mutations were found in 11 patients. There were several new mutations among them: c.544G>A (p.Val182Met), c.1480G>A (p.Ala494Thr), c.1532A>G (p.Lys511Arg) and c. 2156C>T (p.Ala719Val). The authors concluded that the presence of some heterozygous mutations of *NLRP7* in woman may be a risk factor not only for BiCHM, but also for a sporadic mole.

mutated in 14% of patients with recurrent a hydatidiform mole who are negative for *NLRP7*

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139

Sequencing of *KHDC3L* in 97 patients with recurrent moles, reproductive loss and absence of *NLRP7* mutations allows to identify three unrelated patients, each homozygous for one of the two protein-truncating mutations, a novel 4-bp deletion resulting in a frame shift c. 299\_302delTCAA (p.Ile100Argfs\*2), and a previously described 4-bp deletion c.322\_325del‐ GACT (p.Asp108Ilefs\*30), transmitted on a shared haplotype to three patients from different populations [83]. It was also shown that molar tissues from one of the spontaneous abortions were diploid and biparental. In this study, immunofluorescence analysis revealed colocaliza‐ tion of KHDC3L and NLRP7 proteins in lymphoblastoid cell lines from normal subjects. Using cell lines from patients, it was demonstrated that the *KHDC3L* mutations do not change the subcellular localization of protein in haematopoietic cells. This finding highlights the similar‐ ities between the two causative genes for recurrent moles, *KHDC3L* and *NLRP7*, in their subcellular localization, the parental contribution to the mole tissues caused by them, and the presence of several founder mutations and variants in different populations in both of them

It is probable that some patients with imprinting disorders and MLMD also have mutations in two other genes – *NLRP2* (*NALP2*) (19q13.42) and *ZFP57* (6p22.1), which may be involved in imprinting maintenance. The NALP2 is a cytosolic protein of the CATERPILLER's subfam‐ ily. It is suggested that NALP2 is a component of inflammosomes, like NLRP7. Mutation c. 1479delAG (p.Arg493SerfsX32) at the *NLRP2* was found in a family with one healthy child and two children with BWS [84]. *KCNQ1OT1* imprinted centres were hypomethylated in both affected children, whereas *PEG1* hypomethylation was observed in only one of them. Homo‐ zygous mutation at the *NLRP2* was detected in the mother and the affected child, whereas the father, the healthy child and the other diseased child were heterozygotes. The mutation was absent in the control group. This data is in agreement with the hypothesis that *NLRP2* is a maternal-effect gene, like *NLRP7*. It is interesting that *NLRP2 per se*is imprinted. Its monoallelic expression from the maternal chromosome was detected in decidual tissue, foetal heart and liver [85]. However, there is a single report, which does not support the maternal effect of *NLRP2*. In 2013, a paternally inherited mutation c.2077C>T (p.R693W) was observed in BWS patient with *KCNQ1OT1* hypomethylation [61]. This mutation was absent in individuals from the control group and may be pathogenic according to the "PolyPhen-2" database. However,

In 2008, mutations at the *ZNF57* were detected for the first time in seven out of twelve families with TNDM and multiple hypomethylation of imprinted genes *PLAGL1, CRB10, PEG3, KCNQ1OT1, PEG1/MEST*, and *NESPAS* [86]. Homozygous mutation p.C241X was found in a brother with hypomethylation at the *KCNQ1OT1, NASPAS* and *PEG1/MEST* and his sister with *KCNQ1OT1* and *PEG1/MEST* hypomethylation. Proband in another family had deletion c.257\_258delAG (p.E86VfsX28). His father was a heterozygote for this deletion. Compound heterozygous mutation c.683G>A (p.R228H) was detected in two families, including one patient with hypomethylation of *PEG1/MEST* and *NESPAS*. None of the mutations were found

it seems that this example does not cover multiple methylation defects.

mutations.

indicating positive selection and adaptation.

in 200 individuals from the control group.

There are current reports on mutations at almost all exons and introns of *NLRP7* gene, which were associated with BiCHM. The product of the gene belongs to CATERPILLER proteins family. These NLRP proteins are implicated in the activation of proinflammatory caspases through multiprotein complexes called inflammasomes. This gene may act as a feedback regulator of caspase-1-dependent interleukin 1-beta secretion, which is pleiotropic cytokine involved in trophoblast invasion in the uterus during implantation [76].

As mentioned previously, *NLRP7* may be a gene with a maternal effect. The products of such genes are necessary for oocytes to support early embryo development before the activation of embryo genome. These genes do not have an influence on ovulation and fertilization, but the absence of their products leads to the termination of early embryo development. This feature may explain the lack of reproductive problems in *NLRP7* mutation's male carriers. It seems that another gene from the NRLP family in mice – *Nalp5*, is a gene with maternal effect also. *Nalp5-/-* females had normal ovaries. Their oocytes fertilized normally, but embryos arrested their development at the 2-cell stage [77].

NLRP7 protein has no DNA-binding motifs in its sequence, which is why it is unclear how it may be involved in imprinting recording during oogenesis. In this situation, an alternative hypothesis is attractive. According to this hypothesis, the involvement of NLRP7 in BiCHM pathogenesis may be related to its participation in inflammation and autoimmune response. It was found that patients with *NLRP7* mutations cannot provide specific responses to different antigens [78]. As a result, in such women androgenic blastocysts, which are in fact are complete allografts, can implant and develop without rejection from the maternal side. It is also possible that androgenic blastocysts arise spontaneously *de novo* with definite frequency due to errors of fertilization or through epigenetic mechanisms independently from *NRLP7* mutations. In women without *NRLP7* mutations and normal immune systems, such androgenic blastocysts die or stop in development. It is interesting that mutations in other members of the CATER‐ PILLER family – *NLRP1* and *NOD2* were also associated with some clinical forms of vitiligo (MIM #606579) and inflammatory bowel disease (MIM #266600) [78]. There were no reports on the association of molar pregnancies with these autoimmune diseases, except for one study [79]. However, the association of Crohn's disease with recurrent pregnancy loss was noted repeatedly [80, 81]. It is possible that this association is based on common pathogenic mecha‐ nisms involved in the disturbance of autoimmune response regulation.

Mutations at the *NLRP7* gene have not been found in every case of BiCHM and only 48-60% of patients with recurrent hydatidiform moles indicate a heterogeneous nature of this repro‐ ductive pathology. Indeed, in 2011 a type II of BiCHM was described (MIM #614293), which is clinically indistinguishable from the classical variant of the biparental complete mole. It was related to mutations at the *KHDC3L* (*C6orf221*) gene in 6q13. The search for mutations in this gene was performed in 14 pedigrees with BiCHM without *NLRP7* mutations. As a result, homozygous change c.3G>T, deletion c.322\_325delGACT and compound heterozygote c. 322\_325delGACT were found in three families [82]. This study revealed that *KHDC3L* is mutated in 14% of patients with recurrent a hydatidiform mole who are negative for *NLRP7* mutations.

2156C>T (p.Ala719Val). The authors concluded that the presence of some heterozygous mutations of *NLRP7* in woman may be a risk factor not only for BiCHM, but also for a sporadic

There are current reports on mutations at almost all exons and introns of *NLRP7* gene, which were associated with BiCHM. The product of the gene belongs to CATERPILLER proteins family. These NLRP proteins are implicated in the activation of proinflammatory caspases through multiprotein complexes called inflammasomes. This gene may act as a feedback regulator of caspase-1-dependent interleukin 1-beta secretion, which is pleiotropic cytokine

As mentioned previously, *NLRP7* may be a gene with a maternal effect. The products of such genes are necessary for oocytes to support early embryo development before the activation of embryo genome. These genes do not have an influence on ovulation and fertilization, but the absence of their products leads to the termination of early embryo development. This feature may explain the lack of reproductive problems in *NLRP7* mutation's male carriers. It seems that another gene from the NRLP family in mice – *Nalp5*, is a gene with maternal effect also. *Nalp5-/-* females had normal ovaries. Their oocytes fertilized normally, but embryos arrested

NLRP7 protein has no DNA-binding motifs in its sequence, which is why it is unclear how it may be involved in imprinting recording during oogenesis. In this situation, an alternative hypothesis is attractive. According to this hypothesis, the involvement of NLRP7 in BiCHM pathogenesis may be related to its participation in inflammation and autoimmune response. It was found that patients with *NLRP7* mutations cannot provide specific responses to different antigens [78]. As a result, in such women androgenic blastocysts, which are in fact are complete allografts, can implant and develop without rejection from the maternal side. It is also possible that androgenic blastocysts arise spontaneously *de novo* with definite frequency due to errors of fertilization or through epigenetic mechanisms independently from *NRLP7* mutations. In women without *NRLP7* mutations and normal immune systems, such androgenic blastocysts die or stop in development. It is interesting that mutations in other members of the CATER‐ PILLER family – *NLRP1* and *NOD2* were also associated with some clinical forms of vitiligo (MIM #606579) and inflammatory bowel disease (MIM #266600) [78]. There were no reports on the association of molar pregnancies with these autoimmune diseases, except for one study [79]. However, the association of Crohn's disease with recurrent pregnancy loss was noted repeatedly [80, 81]. It is possible that this association is based on common pathogenic mecha‐

Mutations at the *NLRP7* gene have not been found in every case of BiCHM and only 48-60% of patients with recurrent hydatidiform moles indicate a heterogeneous nature of this repro‐ ductive pathology. Indeed, in 2011 a type II of BiCHM was described (MIM #614293), which is clinically indistinguishable from the classical variant of the biparental complete mole. It was related to mutations at the *KHDC3L* (*C6orf221*) gene in 6q13. The search for mutations in this gene was performed in 14 pedigrees with BiCHM without *NLRP7* mutations. As a result, homozygous change c.3G>T, deletion c.322\_325delGACT and compound heterozygote c. 322\_325delGACT were found in three families [82]. This study revealed that *KHDC3L* is

involved in trophoblast invasion in the uterus during implantation [76].

nisms involved in the disturbance of autoimmune response regulation.

their development at the 2-cell stage [77].

mole.

138 Epigenetics and Epigenomics

Sequencing of *KHDC3L* in 97 patients with recurrent moles, reproductive loss and absence of *NLRP7* mutations allows to identify three unrelated patients, each homozygous for one of the two protein-truncating mutations, a novel 4-bp deletion resulting in a frame shift c. 299\_302delTCAA (p.Ile100Argfs\*2), and a previously described 4-bp deletion c.322\_325del‐ GACT (p.Asp108Ilefs\*30), transmitted on a shared haplotype to three patients from different populations [83]. It was also shown that molar tissues from one of the spontaneous abortions were diploid and biparental. In this study, immunofluorescence analysis revealed colocaliza‐ tion of KHDC3L and NLRP7 proteins in lymphoblastoid cell lines from normal subjects. Using cell lines from patients, it was demonstrated that the *KHDC3L* mutations do not change the subcellular localization of protein in haematopoietic cells. This finding highlights the similar‐ ities between the two causative genes for recurrent moles, *KHDC3L* and *NLRP7*, in their subcellular localization, the parental contribution to the mole tissues caused by them, and the presence of several founder mutations and variants in different populations in both of them indicating positive selection and adaptation.

It is probable that some patients with imprinting disorders and MLMD also have mutations in two other genes – *NLRP2* (*NALP2*) (19q13.42) and *ZFP57* (6p22.1), which may be involved in imprinting maintenance. The NALP2 is a cytosolic protein of the CATERPILLER's subfam‐ ily. It is suggested that NALP2 is a component of inflammosomes, like NLRP7. Mutation c. 1479delAG (p.Arg493SerfsX32) at the *NLRP2* was found in a family with one healthy child and two children with BWS [84]. *KCNQ1OT1* imprinted centres were hypomethylated in both affected children, whereas *PEG1* hypomethylation was observed in only one of them. Homo‐ zygous mutation at the *NLRP2* was detected in the mother and the affected child, whereas the father, the healthy child and the other diseased child were heterozygotes. The mutation was absent in the control group. This data is in agreement with the hypothesis that *NLRP2* is a maternal-effect gene, like *NLRP7*. It is interesting that *NLRP2 per se*is imprinted. Its monoallelic expression from the maternal chromosome was detected in decidual tissue, foetal heart and liver [85]. However, there is a single report, which does not support the maternal effect of *NLRP2*. In 2013, a paternally inherited mutation c.2077C>T (p.R693W) was observed in BWS patient with *KCNQ1OT1* hypomethylation [61]. This mutation was absent in individuals from the control group and may be pathogenic according to the "PolyPhen-2" database. However, it seems that this example does not cover multiple methylation defects.

In 2008, mutations at the *ZNF57* were detected for the first time in seven out of twelve families with TNDM and multiple hypomethylation of imprinted genes *PLAGL1, CRB10, PEG3, KCNQ1OT1, PEG1/MEST*, and *NESPAS* [86]. Homozygous mutation p.C241X was found in a brother with hypomethylation at the *KCNQ1OT1, NASPAS* and *PEG1/MEST* and his sister with *KCNQ1OT1* and *PEG1/MEST* hypomethylation. Proband in another family had deletion c.257\_258delAG (p.E86VfsX28). His father was a heterozygote for this deletion. Compound heterozygous mutation c.683G>A (p.R228H) was detected in two families, including one patient with hypomethylation of *PEG1/MEST* and *NESPAS*. None of the mutations were found in 200 individuals from the control group.

Two years later, Mackay and Temple reported about *ZFP57* mutations in 10 out of 16 patients with TNDM and hypomethylation at the *PLAGL1, GRB10* and *PEG3* genes [87]. It is interesting that all patients with maternal hypomethylation syndrome, *ZFP57* mutations and *PLAGL1* hypomethylation also had *PEG3* and *GRB10* hypomethylation. However, in the absence of *ZFP57* mutations, *PEG3* and *GRB10* genes were infrequently affected by methylation defects. This observation indicates that *ZFP57* may be involved in imprinting maintenance in somatic cells in contrast to germinal methylation defects, which are associated with mutations at the *NLRP7*.

single gene basis sometimes modified by parental-of-origin effects. A similar situation is specific for chromatin diseases (ICF, Rett, Rubinshtein-Taybi, Coffin-Lowry, ATR-X syn‐ dromes), which arise due to mutations in genes involved in the control of chromatin organi‐ zation [8]. From this point of view, the presence of one form of TNDM, a classical imprinting disorder, in the OMIM catalogue (MIM # 601410) as a result of *ZFP57* mutations is not

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141

Considering the high incidence of reproductive losses in humans and the elevated level of methylation defects at different imprinted loci in spontaneous abortions, the search for mutations in genes involved in the control of genomic imprinting is a challenge for modern reproductive epigenetics and medicine. In our preliminary study, 11 first trimester missed abortions with MLMD of imprinted genes were tested for the presence of *NLRP7* mutations [92]. Nine genetic variants of *NLRP7* were found. Seven of them were presented in specific databases for women with BiCHM and normal reproductive outcomes. Whereas two new changes, c1405delC and c1444delC, in homozygous form were found in spontaneous abortion with hypomethylation at the *PEG10, KCNQ1, WT1, ZNF215* and hypermethylation at the *INS,*

The reviewed data clearly indicates that epigenetic abnormalities are the leading cause of imprinted gene dysfunction in pregnancy complications and losses. This is not surprising because of the fact that the rate of epimutations is estimated to be one or two orders higher than the incidence of classical gene mutations. It is import to also note that epimutation in one allele is enough to cause the loss of imprinting or the silencing of an imprinted gene due to

The application of genome wide technologies of DNA methylation analysis revealed the phenomenon of multiple methylation defects at imprinting genes both in spontaneous abortions and in some patients with imprinting disorders. Today we are witnesses of data accumulation about spectrum and the incidence of this type of methylation abnormalities in different diseases. However, cautious estimations should be provided because of a lack of current data about possible benign epipolymorphisms of imprinted genes. However, obtained results change and supplement existent concepts about pathogenesis of imprinting disorders. One of the most intriguing findings is that some part of epigenetic imprinting defects has, in fact, a genetic nature due to mutations in genes, which are responsible for imprinting regula‐ tion. This remark may have obvious significance for the likes of molecular genetic diagnosis in the light of the application of high-throughput genomic and post-genomic technologies and for medical genetic counselling. Carriers of mutations in imprinting control genes may have incorrect or instable epigenomes in their gametes or progeny which will be not compatible with fertilization, implantation, normal prenatal development or the delivery of a healthy child. Preimplantation genetic diagnosis for the excluding of embryo transfer with mutations

one of its main inherent features, namely monoallelic expression.

in such genes may be a successful reproductive choice for such couples.

unexpected.

*PWCR1* and *GABRA5* genes.

**7. Conclusion**

*ZFP57* mutations were also observed in patients with other imprinting disorders, like SRS and BWS. For example, seven mutations at the *ZFP57* were found in 30 patients with SRS and hypomethylation of imprinting centre *H19/IGF2* on chromosome 11 [88]. Other imprinted genes were not tested in this study. Six nucleotide changes were recognized as known polymorphisms, whereas one patient had not previously detected homozygous mutation p.R125Q in the exon 6. This mutation was also found in heterozygous form in two of the 80 (2.5%) healthy individuals.

Twenty-seven BWS patients with *KCNQ1OT1* hypomethylation were tested for the presence of *ZFP57* mutations [89]. As a result, three new nucleotide changes were found. Two twin girls and their father were heterozygous for the c.503C>T (p.Ser168Phe). Two other changes c. 723C>T and c.1026T>C were detected in a diseased child and his father.

*ZFP57* mutations were found in a recent study of patients with TNDM, SRS, BWS and methylation defects [61]. New homozygous deletion c.371delC was identified in a patient with TNDM and maternal hypomethylation of five imprinted genes, including *PLAGL1*. Both parents in this family were heterozygous carriers of deletion, whereas it was absent in the 180 individuals from the control group. Two cases of maternal inheritance of the mutation c. 374G>A (p.R125Q) were observed in a SRS patient with *H19/IGF2* hypomethylation and in a BWS patient with a loss of methylation at the *KCNQ1OT1*. This mutation was not found in the control sample. The mother of the SRS patient was a compound heterozygote for p.R125Q and c.559G>A (p.R187C) mutation.

The protein encoded by *ZFP57* is a zinc finger protein containing the Kruppel-associated box repressor (KRAB) domain, which acts as transcription repressor. It is interesting that this domain may be involved in the *de novo* DNA methylation during mouse embryogenesis [90]. It was also shown that *Zfp57* mutations in mice may induce multiple methylation defects of imprinted genes. Partial hypomethylation of maternal and paternal alleles was noted in progeny with *Zfp57-/-* genotype in zygote [Li et al., 2008]. The authors of this study suggested that *Zfp57* is a maternal-zygotic effect gene and its product is required for imprinting mainte‐ nance in different genomic loci.

Taking the discussed results together, it is possible to make an unexpected conclusion that some part of imprinting diseases and reproductive disorders associated with abnormal imprinting are related to defects in gene (or genes) involved in the establishment and main‐ tenance of epigenetic organization of imprinted loci. In other words, imprinting diseases, or at least some part of them, that were usually considered epigenetic in nature, have, in fact, a single gene basis sometimes modified by parental-of-origin effects. A similar situation is specific for chromatin diseases (ICF, Rett, Rubinshtein-Taybi, Coffin-Lowry, ATR-X syn‐ dromes), which arise due to mutations in genes involved in the control of chromatin organi‐ zation [8]. From this point of view, the presence of one form of TNDM, a classical imprinting disorder, in the OMIM catalogue (MIM # 601410) as a result of *ZFP57* mutations is not unexpected.

Considering the high incidence of reproductive losses in humans and the elevated level of methylation defects at different imprinted loci in spontaneous abortions, the search for mutations in genes involved in the control of genomic imprinting is a challenge for modern reproductive epigenetics and medicine. In our preliminary study, 11 first trimester missed abortions with MLMD of imprinted genes were tested for the presence of *NLRP7* mutations [92]. Nine genetic variants of *NLRP7* were found. Seven of them were presented in specific databases for women with BiCHM and normal reproductive outcomes. Whereas two new changes, c1405delC and c1444delC, in homozygous form were found in spontaneous abortion with hypomethylation at the *PEG10, KCNQ1, WT1, ZNF215* and hypermethylation at the *INS, PWCR1* and *GABRA5* genes.

## **7. Conclusion**

Two years later, Mackay and Temple reported about *ZFP57* mutations in 10 out of 16 patients with TNDM and hypomethylation at the *PLAGL1, GRB10* and *PEG3* genes [87]. It is interesting that all patients with maternal hypomethylation syndrome, *ZFP57* mutations and *PLAGL1* hypomethylation also had *PEG3* and *GRB10* hypomethylation. However, in the absence of *ZFP57* mutations, *PEG3* and *GRB10* genes were infrequently affected by methylation defects. This observation indicates that *ZFP57* may be involved in imprinting maintenance in somatic cells in contrast to germinal methylation defects, which are associated with mutations at the

*ZFP57* mutations were also observed in patients with other imprinting disorders, like SRS and BWS. For example, seven mutations at the *ZFP57* were found in 30 patients with SRS and hypomethylation of imprinting centre *H19/IGF2* on chromosome 11 [88]. Other imprinted genes were not tested in this study. Six nucleotide changes were recognized as known polymorphisms, whereas one patient had not previously detected homozygous mutation p.R125Q in the exon 6. This mutation was also found in heterozygous form in two of the 80

Twenty-seven BWS patients with *KCNQ1OT1* hypomethylation were tested for the presence of *ZFP57* mutations [89]. As a result, three new nucleotide changes were found. Two twin girls and their father were heterozygous for the c.503C>T (p.Ser168Phe). Two other changes c.

*ZFP57* mutations were found in a recent study of patients with TNDM, SRS, BWS and methylation defects [61]. New homozygous deletion c.371delC was identified in a patient with TNDM and maternal hypomethylation of five imprinted genes, including *PLAGL1*. Both parents in this family were heterozygous carriers of deletion, whereas it was absent in the 180 individuals from the control group. Two cases of maternal inheritance of the mutation c. 374G>A (p.R125Q) were observed in a SRS patient with *H19/IGF2* hypomethylation and in a BWS patient with a loss of methylation at the *KCNQ1OT1*. This mutation was not found in the control sample. The mother of the SRS patient was a compound heterozygote for p.R125Q and

The protein encoded by *ZFP57* is a zinc finger protein containing the Kruppel-associated box repressor (KRAB) domain, which acts as transcription repressor. It is interesting that this domain may be involved in the *de novo* DNA methylation during mouse embryogenesis [90]. It was also shown that *Zfp57* mutations in mice may induce multiple methylation defects of imprinted genes. Partial hypomethylation of maternal and paternal alleles was noted in progeny with *Zfp57-/-* genotype in zygote [Li et al., 2008]. The authors of this study suggested that *Zfp57* is a maternal-zygotic effect gene and its product is required for imprinting mainte‐

Taking the discussed results together, it is possible to make an unexpected conclusion that some part of imprinting diseases and reproductive disorders associated with abnormal imprinting are related to defects in gene (or genes) involved in the establishment and main‐ tenance of epigenetic organization of imprinted loci. In other words, imprinting diseases, or at least some part of them, that were usually considered epigenetic in nature, have, in fact, a

723C>T and c.1026T>C were detected in a diseased child and his father.

*NLRP7*.

140 Epigenetics and Epigenomics

(2.5%) healthy individuals.

c.559G>A (p.R187C) mutation.

nance in different genomic loci.

The reviewed data clearly indicates that epigenetic abnormalities are the leading cause of imprinted gene dysfunction in pregnancy complications and losses. This is not surprising because of the fact that the rate of epimutations is estimated to be one or two orders higher than the incidence of classical gene mutations. It is import to also note that epimutation in one allele is enough to cause the loss of imprinting or the silencing of an imprinted gene due to one of its main inherent features, namely monoallelic expression.

The application of genome wide technologies of DNA methylation analysis revealed the phenomenon of multiple methylation defects at imprinting genes both in spontaneous abortions and in some patients with imprinting disorders. Today we are witnesses of data accumulation about spectrum and the incidence of this type of methylation abnormalities in different diseases. However, cautious estimations should be provided because of a lack of current data about possible benign epipolymorphisms of imprinted genes. However, obtained results change and supplement existent concepts about pathogenesis of imprinting disorders. One of the most intriguing findings is that some part of epigenetic imprinting defects has, in fact, a genetic nature due to mutations in genes, which are responsible for imprinting regula‐ tion. This remark may have obvious significance for the likes of molecular genetic diagnosis in the light of the application of high-throughput genomic and post-genomic technologies and for medical genetic counselling. Carriers of mutations in imprinting control genes may have incorrect or instable epigenomes in their gametes or progeny which will be not compatible with fertilization, implantation, normal prenatal development or the delivery of a healthy child. Preimplantation genetic diagnosis for the excluding of embryo transfer with mutations in such genes may be a successful reproductive choice for such couples.

## **Author details**

I.N. Lebedev\*

Address all correspondence to: igor.lebedev@medgenetics.ru

Institute of Medical Genetics, Tomsk, Russia

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[78] Deveault C., Qian J.H., Chebaro W.A. et al., NLRP7 mutations in women with dip‐ loid androgenetic and triploid moles: a proposed mechanism for mole formation.

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form moles. *Molecular Human Reproduction* 2008; 14(1) 33-40.

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[54] Arima T., Kamikihara T., Hayashida T. et al., ZAC, LIT1 (KCNQ1OT1) and p57KIP2 (CDKN1C) are in an imprinted gene network that may play a role in Beckwith-Wie‐

[55] Mackay D.J.G., Hahnemann J.M., Boonen S.E. et al., Epimutation of the TNDM locus and the Beckwith-Wiedemann syndrome centromeric locus in individuals with tran‐

[56] Mackay D.J.G., Boonen S.E., Clayton-Smith J. et al., A maternal hypomethylation syndrome presenting as transient neonatal diabetes mellitus. *Human Genetics* 2006;

[57] Lim D., Bowdin S., Tee L. et al., Clinical and molecular genetic features of Beckwith-Wiedemann syndrome associated with assisted reproductive technologies. *Human*

[58] Yamazawa K., Kagami M., Nagai T. et al., Molecular and clinical findings and their correlations in Silver-Russell syndrome: implications for a positive role of *IGF2* in growth determination and differential imprinting regulation of the IGF2-H19 do‐ main in bodies and placentas. *Journal of Molecular Medicine* 2008; 86(10) 1171-1181. [59] Bartholdi D., Krajewska-Walasek M., Ounap K. et al., Epigenetic mutations of the im‐ printed IGF2-H19 domain in Silver-Russell syndrome (SRS): results from a large co‐ hort of patients with SRS and SRS-like phenotypes. *Journal of Medical Genetics* 2009;

[60] Baple E.L., Poole R.L., Mansour S et al., An atypical case of hypomethylation at mul‐ tiple imprinted loci. *European Journal of Human Genetics* 2011; 19(3) 360-362.

[61] Court F., Martin-Trujillo A., Romanelli V. et al., Genome-wide allelic mathylation analysis reveals disease-specific susceptibility to multiple methylation defects in im‐

[62] Rossignol S., Steunou V., Chalas C. et al., The epigenetic imprinting defect of patients with Beckwith–Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region. *Journal of Medical Genetics* 2006; 43(12) 902-907. [63] Bliek J., Verde G., Callaway J. et al., Hypomethylation at multiple maternally methy‐ lated imprinted regions including PLAGL1 and GNAS loci in Beckwith–Wiedemann

[64] Azzi S., Rossignol S., Steunou V. et al., Multilocus methylation analysis in a large co‐ hort of 11p15-related foetal growth disorders (Russell Silver and Beckwith Wiede‐ mann syndromes) reveals simultaneous loss of methylation at paternal and maternal

[65] Bliek J., Alders M., Maas S.M. et al., Lessons from BWS twins: complex maternal and paternal hypomethylation and a common source of haematopoietic stem cells. *Euro‐*

printing syndromes. *Human Mutation* 2013; 34(4) 595-602.

syndrome. *European Journal of Human Genetics* 2009; 17(5) 611-619.

imprinted loci. *Human Molecular Genetics* 2009; 18(24) 4724-4733.

*pean Journal of Human Genetics* 2009; 17(12) 1625-1634.

sient neonatal diabetes mellitus. *Human Genet*ics 2006; 119(1-2) 179–184.

demann syndrome. *Nucleic Acids Research* 2005; 33(8) 2650–2660.

120(2) 262–269.

146 Epigenetics and Epigenomics

46(3) 192-197.

*Reproduction* 2009; 24(3) 741-747.


[79] Hovdenak N., Ulcerative colitis, vitiligo and hydatidiform mole. *Tidsskrift for Den Norske Legeforening* 1979; 20(23) 1089-1090.

**Chapter 6**

**Methylomes**

Minghua Wu

**1. Introduction**

http://dx.doi.org/10.5772/57535

Additional information is available at the end of the chapter

Epigenetic modifications stably influence gene expression without changing the underlying DNA sequence [1]. The epigenomic era has revealed a well-connected network of molecular processes. These processes comprise abnormal methylomes, transcriptosomes, genome-wide histone post-transcriptional modifications patterns, histone variants, and noncoding RNAs [2]. The genome-wide DNA methylation status of cells exists in a methylated, hydroxymethylated or unmethylated state, collectively referred to as the DNA methylome [3]. Methylation of the DNA (DNAme) occurs in position 5 in cytosine residues. In mammals, the vast majority (98%) of DNA methylation occurs in CpG (cytosine-phosphate-guanine) dinucleotides in somatic cells [4]. In embryomic stem (ES) cells, however, about one-quarter of all DNA methylation occurs in non-CpG context [4]. The haploid human genome contains approximately 29 million CpGs, DNA methylation involves the transfer of a methyl group to cytosine in a CpG dinu‐ cleotide through DNA methyltransferases that creates or maintains methylation patterns [3]. Hydroxymethylation of cytosines has also been reported, though its biological functions are unknown. Several methods have been developed which enable capture of genome-wide profiling of DNA methylation. The complete DNA methylomes for several organisms are now available, helping clarify the evolutionary story of this epigenetic mark and its distribution in key genomic elements. The variation of DNA methylome plays an important role in regulating normal development and differentiation. DNA methylation patterns can be inherited and influenced by the environment, diet and aging, and disrupted in diseases [5]. From a functional perspective, the DNA methylome variation is a stable change in a transcriptional regulatory element, which changes the expression of a gene without any change in DNA sequence or in the intracellular environment. However, a change in environmental conditions could perturb the stability profile of the methylome at a locus, changing the probability of variant states

arising, even making a variant state more stable than the previous reference state [6].

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


## **Chapter 6**

## **Methylomes**

[79] Hovdenak N., Ulcerative colitis, vitiligo and hydatidiform mole. *Tidsskrift for Den*

[80] Gasbarrini A., Torre E.S., Trivellini C. et al., Recurrent spontaneous abortion and in‐ trauterine fetal growth retardation as symptoms of coeliac disease. *Lancet* 2000;

[81] Lejeune V. Early recurrent spontaneous abortion: How to take care in 2006? //*Gynéco‐*

[82] Parry D.A., Logan C.V., Hayward B.E. et al., Mutations causing familial biparental hydatidiform mole implicate *c6orf221* as a possible regulator of genomic imprinting

[83] Reddy R., Akoury E., Nguyen N.M.P. et al., Report of four new patients with pro‐ tein-truncating mutations in *C6orf221/KHDC3L* and colocolization with *NLRP7*. *Euro‐*

[84] Meyer E., Lim D., Pasha S. et al., Germline mutation in *NLRP2* (*NALP2*) in a familial imprinting disorder (Beckwith-Wiedemann syndrome). *PloS Genetics* 2009; 5(3)

[85] Bjornsson H.T., Albert T.J., Ladd-Acosta C.M. et al., SNP-specific array-based allele-

[86] Mackay D.J., Callaway J.L., Marks S.M. et al., Hypomethylation of multiple imprint‐ ed loci in individuals with transient neonatal diabetes is associated with mutations in

[87] Mackay D.J., Temple I.K.. Transient neonatal diabetes mellitus type 1. *American Jour‐ nal of Medical Genetics Part C Seminars in Medical Genetics* 2010; 154(3) 335-342.

[88] Spengler S., Gogiel M., Schönherr N. et al., Screening for genomic variants in ZFP57 in Silver-Russell syndrome patients with 11p15 epimutations. *European Journal of*

[89] Boonen S.E., Hahnemann J.M., Mackay D.J. et al., No evidence for pathogenic var‐ iants or maternal effect of ZFP57 as the cause of Beckwith-Wiedemann Syndrome.

[90] Wiznerowicz M., Jakobsson J., Szulc J. et al., The Kruppel-associated box repressor domain can trigger *de novo* promoter methylation during mouse early embryogene‐

[91] Li X., Ito M., Zhou F. et al., A maternal-zygotic effect gene, *Zfp57*, maintains both ma‐

[92] Lepshin M.V., Sazhenova E.A., Lebedev I.N.. Search for NLRP7 mutations in first tri‐ mester missed abortions with multiple methylation changes in imprinted genes. *Eu‐*

specific expression analysis. *Genome Research* 2008; 18(5) 771-779.

in the human oocyte. *American Journal of Human Genetics* 2011; 89(3) 451-458.

*Norske Legeforening* 1979; 20(23) 1089-1090.

*logie Obstétrique & Fertilite* 2006;34(10) 927-937.

*pean Journal of Human Genetics* 2013; 21(9) 957-964.

e1000423 doi: 10.1371/journal.pgen.1000423.

*ZFP57*. *Nature Genetics* 2008; 40(8) 949-951.

*Medical Genetics* 2009; 52(6) 415-416.

*European Journal of Human Genetics* 2012; 20(1) 119-121.

sis. *Journal of Biological Chemistry* 2007; 282(47) 34535-34541.

*ropean Journal of Human Genetics* 2013; 21(Supplement 2) 588.

ternal and paternal imprints. *Developmental Cell* 2008; 15(4) 547-557.

356(9227) 399-400.

148 Epigenetics and Epigenomics

Minghua Wu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57535

## **1. Introduction**

Epigenetic modifications stably influence gene expression without changing the underlying DNA sequence [1]. The epigenomic era has revealed a well-connected network of molecular processes. These processes comprise abnormal methylomes, transcriptosomes, genome-wide histone post-transcriptional modifications patterns, histone variants, and noncoding RNAs [2]. The genome-wide DNA methylation status of cells exists in a methylated, hydroxymethylated or unmethylated state, collectively referred to as the DNA methylome [3]. Methylation of the DNA (DNAme) occurs in position 5 in cytosine residues. In mammals, the vast majority (98%) of DNA methylation occurs in CpG (cytosine-phosphate-guanine) dinucleotides in somatic cells [4]. In embryomic stem (ES) cells, however, about one-quarter of all DNA methylation occurs in non-CpG context [4]. The haploid human genome contains approximately 29 million CpGs, DNA methylation involves the transfer of a methyl group to cytosine in a CpG dinu‐ cleotide through DNA methyltransferases that creates or maintains methylation patterns [3]. Hydroxymethylation of cytosines has also been reported, though its biological functions are unknown. Several methods have been developed which enable capture of genome-wide profiling of DNA methylation. The complete DNA methylomes for several organisms are now available, helping clarify the evolutionary story of this epigenetic mark and its distribution in key genomic elements. The variation of DNA methylome plays an important role in regulating normal development and differentiation. DNA methylation patterns can be inherited and influenced by the environment, diet and aging, and disrupted in diseases [5]. From a functional perspective, the DNA methylome variation is a stable change in a transcriptional regulatory element, which changes the expression of a gene without any change in DNA sequence or in the intracellular environment. However, a change in environmental conditions could perturb the stability profile of the methylome at a locus, changing the probability of variant states arising, even making a variant state more stable than the previous reference state [6].

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Methylomes and evolution**

DNA methylation is a typical characteristic of most eukaryotes and some of its features are conserved in many species. The methylation states that present in the germline are heritable and participate in evolution. Boffelli D and Martin DI [6] combined phylogenomic and somatic methylation data to infer germline methylation states. Methylated CGs undergo mutation to TG much more frequently than unmethylated CGs, but only CG decay that occurs in the germline results in heritable sequence changes that can become fixed within a species. The predominant trend within the genome is to lose methylated cytosines and this destrucrion of a CpG dinucleotide by a SNP has been shown to lead to significant cis-methylation effects [7]. The loss and gain of CpGs over time is proposed to be a significant evolutionary device, the polymorphic nature of CpG-SNP dinucleotide sequences will help define human population epigenomics [8].

Currently, by the base-resolution sequencing, the complete methylomes for 25 organisms are available (10 animals, 8 plants, 2 insect and 5 fungi). These eukaryotic methylomes have provided initial insights into the evolutionary history of DNA methylation (Figure 1). Since the density of possible methylation sites, and the distribution of 5-methyl-cytosines (5meC) is not uniform in the genome contexts, the role of DNA methylation in promoters, gene bodies, regulatory features, and transposable and repetitive elements can be remarkably different. Such as, the evolutionary history of DNA methylation in gene bodies and transposons is independent. Zemach et al. quantified DNA methylation in 17 eukaryotic genomes and found that gene body methylation is an ancient property of eukaryotic genomes, and is conserved between plants and animals, whereas selective methylation of transposons is not [9]. The transposon methylation is only conserved in fungi [10], and appears to be related to the degree of sexual outcrossing [11]. In general, the DNA methylation landscape can be either continuous along the genome, or constituted by a series of heavily methylated DNA domains interspersed with domains that are methylation free, and the methylation pattern is quite conserved [5].

## **3. Human methylomes**

Because of its association with human development and disease, DNA methylation has stayed in the research focus for almost half a century. The development of methods for whole-genome methylation profiling has now enabled acquisition of the complete human methylomes, especially powerful are methylome profiling techniques with single-base resolution sequenc‐ ing. Genome-wide, tissue-specific or cell type-specific DNA methylation profiling has begun to shift the focus of DNA methylation research from mostly promoters and immediate upstream enhancers to including intragenic regions and distal intergenetic regions [12]. These revealed that methylation of gene bodies is more frequent than in promoters in the vertebrate genomes [13]. Shen et al [14] characterized the methylome in purified peripheral blood monocytes (PBMs) by using methylated DNA immunoprecipitation combined with highthroughput sequencing, and found that promoters were commonly (58%) found to be

unmethylated; whereas protein coding regions were largely (84%) methylated. Zilbauer et al [15] provide detailed functional genome-wide methylome maps of five primary peripheral blood leukocyte subsets including T-cells, B-cells, monocytes/ macrophages and neutrophils obtained from healthy individuals and identified important cell-type specific hypomethylated regions (HMRs) that strongly correlate with gene transcription levels. SNPs associated with

being A, C or T).

**Figure 1.** Methylation levels in 25 eukaryotic organisms. The organisms are organized according to their evolutionary distance. Tree topology is determined from the NCBI Taxonomy (http://www.ncbi.nlm.nih.gov/guide/taxonomy/) and displayed using TreeView X (http://darwin.zoology.gla.ac.uk/~rpage/treeviewx/index.html). The genome size is indicated together with the percentage of methylated sites within three sequence contexts: CpG, CHG and CHH (H

Methylomes

151

http://dx.doi.org/10.5772/57535


**2. Methylomes and evolution**

epigenomics [8].

150 Epigenetics and Epigenomics

**3. Human methylomes**

DNA methylation is a typical characteristic of most eukaryotes and some of its features are conserved in many species. The methylation states that present in the germline are heritable and participate in evolution. Boffelli D and Martin DI [6] combined phylogenomic and somatic methylation data to infer germline methylation states. Methylated CGs undergo mutation to TG much more frequently than unmethylated CGs, but only CG decay that occurs in the germline results in heritable sequence changes that can become fixed within a species. The predominant trend within the genome is to lose methylated cytosines and this destrucrion of a CpG dinucleotide by a SNP has been shown to lead to significant cis-methylation effects [7]. The loss and gain of CpGs over time is proposed to be a significant evolutionary device, the polymorphic nature of CpG-SNP dinucleotide sequences will help define human population

Currently, by the base-resolution sequencing, the complete methylomes for 25 organisms are available (10 animals, 8 plants, 2 insect and 5 fungi). These eukaryotic methylomes have provided initial insights into the evolutionary history of DNA methylation (Figure 1). Since the density of possible methylation sites, and the distribution of 5-methyl-cytosines (5meC) is not uniform in the genome contexts, the role of DNA methylation in promoters, gene bodies, regulatory features, and transposable and repetitive elements can be remarkably different. Such as, the evolutionary history of DNA methylation in gene bodies and transposons is independent. Zemach et al. quantified DNA methylation in 17 eukaryotic genomes and found that gene body methylation is an ancient property of eukaryotic genomes, and is conserved between plants and animals, whereas selective methylation of transposons is not [9]. The transposon methylation is only conserved in fungi [10], and appears to be related to the degree of sexual outcrossing [11]. In general, the DNA methylation landscape can be either continuous along the genome, or constituted by a series of heavily methylated DNA domains interspersed with domains that are methylation free, and the methylation pattern is quite conserved [5].

Because of its association with human development and disease, DNA methylation has stayed in the research focus for almost half a century. The development of methods for whole-genome methylation profiling has now enabled acquisition of the complete human methylomes, especially powerful are methylome profiling techniques with single-base resolution sequenc‐ ing. Genome-wide, tissue-specific or cell type-specific DNA methylation profiling has begun to shift the focus of DNA methylation research from mostly promoters and immediate upstream enhancers to including intragenic regions and distal intergenetic regions [12]. These revealed that methylation of gene bodies is more frequent than in promoters in the vertebrate genomes [13]. Shen et al [14] characterized the methylome in purified peripheral blood monocytes (PBMs) by using methylated DNA immunoprecipitation combined with highthroughput sequencing, and found that promoters were commonly (58%) found to be

**Figure 1.** Methylation levels in 25 eukaryotic organisms. The organisms are organized according to their evolutionary distance. Tree topology is determined from the NCBI Taxonomy (http://www.ncbi.nlm.nih.gov/guide/taxonomy/) and displayed using TreeView X (http://darwin.zoology.gla.ac.uk/~rpage/treeviewx/index.html). The genome size is indicated together with the percentage of methylated sites within three sequence contexts: CpG, CHG and CHH (H being A, C or T).

unmethylated; whereas protein coding regions were largely (84%) methylated. Zilbauer et al [15] provide detailed functional genome-wide methylome maps of five primary peripheral blood leukocyte subsets including T-cells, B-cells, monocytes/ macrophages and neutrophils obtained from healthy individuals and identified important cell-type specific hypomethylated regions (HMRs) that strongly correlate with gene transcription levels. SNPs associated with immune-mediated disease in genome-wide association studies (GWAS) preferentially localised to these cell-specific regulatory HMRs, offering insight into the pathogenesis role of DNA hypomethylation in regulating immune mediated disease. Recent insights into tissuespecific intra-and intergenic methylation and into cancer methylomes suggest that both cancerassociated DNA hypomethylation and hypermethylation are found throughout the genome [16]. The hypermethylation includes promoters of tumor suppressor genes whose expression becomes repressed, thereby facilitating cancer formation. Cancer-associated DNA hypome‐ thylation from intergenic enhancers, promoter regions, silencers, and chromatin boundary elements may alter transcription rates. Whereas, the intragenic DNA hypomethylation might modulate alternative promoter usage, production of intragenic noncoding RNA transcripts, cotranscriptional splicing, and transcription initiation or elongation [16].The new discoveries that genomic 5-hydroxymethylcytosine is an intermediate in DNA demethylation and exhibits cancer-associated losses.

tency but not self-renewal in ESCs. Embryonic stem cells deficient in Dnmt1 and/or Dnmt3a/3b or lacking CpG-binding proteins show a loss of pluripotency and severe impair‐ ment of differentiation potential, but still maintain self-renew [20-23]. (2) CpG methylation contributes to differentiation of ESCs. In a search for differentially methylated (DM) regions (DMRs) by reduced-representation bisulfite sequencing(RRBS), Meissner et al [24] found that approximately 8% of CpGs that were unmethylated in ESCs became methylated in ESCderived neural progenitor cells and approximately 2% of CpGs methylated in ESCs were unmethylated in the neural progenitor derivatives; Genomic analysis provides supporting evidence for the CpG methylation of gene promoters to selectively silence differentiation genes in ESCs, and global DNA demethylation is mostly linked with the upregulation of tissuespecific genes [25]. (3) ESCs are enriched in non-CpG methylation. A recent major study using Methyl-Seq technology reports significant non-CpG methylation in human ESCs, estimating nearly 25% of total cytosine methylation to be non-CpG sites, with CHG and CHH as the major motifs (where H=A, C, or T). Genomic regions enriched in non-CpG methylation are associated with genes involved in RNA processing, RNA splicing and RNA metabolic processes. Interestingly, enrichment of non-CpG methylation in gene bodies correlates with significantly

Methylomes

153

http://dx.doi.org/10.5772/57535

Multipotent stem cells provide a unique intermediate between pluripotent ESCs and unipotent differentiated cells. Multipotent stem cells also show significant reprogramming variability,

Mesenchymal stem cells (MSCs) are multipotent stem cells of mesodermal origin that can be isolated from various sources and induced into different cell types [27]. Adipose tissue-derived stem cells (ADSCs) are isolated from liposuction material, which provide an abundant source of MSCs. Studies show that select adipogenic and nonadipogenic promoters in MSCs, ADSCs and hESCs are hypomethylated and hypermethylated, respectively, suggesting DNA meth‐ ylation controls adipogenic differentiation by activating adipogenic-related genes and silencing nonspecific lineage genes[27]. In addition, epigenomic changes in DNA methylation and chromatin structure have been hypothesized to be critical in the determination of lineagespecific differentiation and tumorigenesis of MSCs [27]. Leu et al [27] applied a targeted DNA methylation method to methylate a polycomb group protein-governed gene, Trip10, in MSCs, which accelerated the cell fate determination of MSCs. However, targeted methylation of HIC1 and RassF1A, both tumor suppressor genes, transformed MSCs into tumor stem cell-like cells.

Hematopoietic stem cells (HSCs) are a special population of multipotent stem cells that are derived from the bone marrow and give rise to a subset of mature blood cells that directs all the immune responses. Accumulating evidence suggests that DNA methylation play critical roles in the maintenance of both self-renewing hematopoietic stem cells and leukemic

including somatic memory and aberrant reprogramming of DNA methylation [26].

more intronic RNA.

*4.3.1. Mesenchymal stem cells*

*4.3.2. Hematopoietic stem cells*

**4.3. DNA methylation remodeling in multipotent stem cells**

## **4. Methylomes in cell differentiation and reprogramming**

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state [17].That is, cell differentiation is a process characterized by the progressive loss of developmental potential and gain in functional specialization. During this process, DNA methylation plays an important role in epigenetic programming by silencing developmental genes and activating tissue-specific genes, thus establishing a cellular memory that defines both cell lineage and cell type.

#### **4.1. DNA methylation remodeling in the embryo development**

Over the course of mammalian development, the genome undergo nearly complete remodel‐ ing of DNA methylation patterns. Primordial germ cells begin with very low DNA methylation levels, then with gametogenesis parental imprinting tags are established, with substantially methylated but differing methylomes in the sperm and egg. In the preimplantation early embryo there is a wave of genome-wide demethylation that occurs, which is rapid in the paternal genome, except for centromeric, repetitive and paternally imprinted genes, with a comparative slow process occurring in the maternal genome [18].This is then followed by heavy de novo methylation, DNA methylation patterns are then progressively re-established, marking gradual commitment towards lineage-specific differentiation [19].

#### **4.2. DNA methylomes in embryonic stem cells**

Embryonic stem cells (ESCs) are a special population of pluripotent cells derived from the inner cell mass (ICM) of a blastocyst during mammalian development. ESCs retain the ability to indefinitely self-renew and differentiate into all cell types found in the adult body. The importance of DNA methylation in ESC stemness maintenance and differentiation is indicated by diverse studies demonstrating the following: (1) DNA methylation is essential for pluripo‐ tency but not self-renewal in ESCs. Embryonic stem cells deficient in Dnmt1 and/or Dnmt3a/3b or lacking CpG-binding proteins show a loss of pluripotency and severe impair‐ ment of differentiation potential, but still maintain self-renew [20-23]. (2) CpG methylation contributes to differentiation of ESCs. In a search for differentially methylated (DM) regions (DMRs) by reduced-representation bisulfite sequencing(RRBS), Meissner et al [24] found that approximately 8% of CpGs that were unmethylated in ESCs became methylated in ESCderived neural progenitor cells and approximately 2% of CpGs methylated in ESCs were unmethylated in the neural progenitor derivatives; Genomic analysis provides supporting evidence for the CpG methylation of gene promoters to selectively silence differentiation genes in ESCs, and global DNA demethylation is mostly linked with the upregulation of tissuespecific genes [25]. (3) ESCs are enriched in non-CpG methylation. A recent major study using Methyl-Seq technology reports significant non-CpG methylation in human ESCs, estimating nearly 25% of total cytosine methylation to be non-CpG sites, with CHG and CHH as the major motifs (where H=A, C, or T). Genomic regions enriched in non-CpG methylation are associated with genes involved in RNA processing, RNA splicing and RNA metabolic processes. Interestingly, enrichment of non-CpG methylation in gene bodies correlates with significantly more intronic RNA.

#### **4.3. DNA methylation remodeling in multipotent stem cells**

Multipotent stem cells provide a unique intermediate between pluripotent ESCs and unipotent differentiated cells. Multipotent stem cells also show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation [26].

#### *4.3.1. Mesenchymal stem cells*

immune-mediated disease in genome-wide association studies (GWAS) preferentially localised to these cell-specific regulatory HMRs, offering insight into the pathogenesis role of DNA hypomethylation in regulating immune mediated disease. Recent insights into tissuespecific intra-and intergenic methylation and into cancer methylomes suggest that both cancerassociated DNA hypomethylation and hypermethylation are found throughout the genome [16]. The hypermethylation includes promoters of tumor suppressor genes whose expression becomes repressed, thereby facilitating cancer formation. Cancer-associated DNA hypome‐ thylation from intergenic enhancers, promoter regions, silencers, and chromatin boundary elements may alter transcription rates. Whereas, the intragenic DNA hypomethylation might modulate alternative promoter usage, production of intragenic noncoding RNA transcripts, cotranscriptional splicing, and transcription initiation or elongation [16].The new discoveries that genomic 5-hydroxymethylcytosine is an intermediate in DNA demethylation and exhibits

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state [17].That is, cell differentiation is a process characterized by the progressive loss of developmental potential and gain in functional specialization. During this process, DNA methylation plays an important role in epigenetic programming by silencing developmental genes and activating tissue-specific genes, thus establishing a cellular memory

Over the course of mammalian development, the genome undergo nearly complete remodel‐ ing of DNA methylation patterns. Primordial germ cells begin with very low DNA methylation levels, then with gametogenesis parental imprinting tags are established, with substantially methylated but differing methylomes in the sperm and egg. In the preimplantation early embryo there is a wave of genome-wide demethylation that occurs, which is rapid in the paternal genome, except for centromeric, repetitive and paternally imprinted genes, with a comparative slow process occurring in the maternal genome [18].This is then followed by heavy de novo methylation, DNA methylation patterns are then progressively re-established,

Embryonic stem cells (ESCs) are a special population of pluripotent cells derived from the inner cell mass (ICM) of a blastocyst during mammalian development. ESCs retain the ability to indefinitely self-renew and differentiate into all cell types found in the adult body. The importance of DNA methylation in ESC stemness maintenance and differentiation is indicated by diverse studies demonstrating the following: (1) DNA methylation is essential for pluripo‐

**4. Methylomes in cell differentiation and reprogramming**

**4.1. DNA methylation remodeling in the embryo development**

marking gradual commitment towards lineage-specific differentiation [19].

cancer-associated losses.

152 Epigenetics and Epigenomics

that defines both cell lineage and cell type.

**4.2. DNA methylomes in embryonic stem cells**

Mesenchymal stem cells (MSCs) are multipotent stem cells of mesodermal origin that can be isolated from various sources and induced into different cell types [27]. Adipose tissue-derived stem cells (ADSCs) are isolated from liposuction material, which provide an abundant source of MSCs. Studies show that select adipogenic and nonadipogenic promoters in MSCs, ADSCs and hESCs are hypomethylated and hypermethylated, respectively, suggesting DNA meth‐ ylation controls adipogenic differentiation by activating adipogenic-related genes and silencing nonspecific lineage genes[27]. In addition, epigenomic changes in DNA methylation and chromatin structure have been hypothesized to be critical in the determination of lineagespecific differentiation and tumorigenesis of MSCs [27]. Leu et al [27] applied a targeted DNA methylation method to methylate a polycomb group protein-governed gene, Trip10, in MSCs, which accelerated the cell fate determination of MSCs. However, targeted methylation of HIC1 and RassF1A, both tumor suppressor genes, transformed MSCs into tumor stem cell-like cells.

#### *4.3.2. Hematopoietic stem cells*

Hematopoietic stem cells (HSCs) are a special population of multipotent stem cells that are derived from the bone marrow and give rise to a subset of mature blood cells that directs all the immune responses. Accumulating evidence suggests that DNA methylation play critical roles in the maintenance of both self-renewing hematopoietic stem cells and leukemic stem cells [29]. Changes in the DNA methylation profile have a critical role in the divi‐ sion of these stem cells into the myeloid and lymphoid lineages and in the establishment of a specific phenotype and functionality in each terminally differentiated cell type [30]. HSCs deficient in both Dnmt3a and 3b show a loss of proliferative ability but retain differentiation potential, suggesting de novo methylation is important for self-renewal in HSCs [28]. The aberrant DNA methylation has been associated with several immune deficiencies and autoimmune disorders [30].

*4.4.2. Non-CpG cytosine methylation in cells reprogramming*

**5. Methylomes in cancer**

Although methylation mainly occurs on the cytosines in the CpG dinucleotide context, non-CG methylation (mCH:DNA methylation targeting CpA, CpT, and CpC dinucleotides) is prevalent in brain, oocytes and pluripotent stem cell [38-40]. Compared to non-growing oocytes (NGOs), germinal vesicle oocytes (GVOs) were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome[38]. Shirane et al [39] found that nearly two-thirds of all meth‐ ylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Ziller et al [40] reported a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types, and confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Non-CpG cytosine methylation has been identified at a high level in stem cells and reprogrammed progenitor cells, indicating that loss of this form of methylation may be critical in the path from pluripotency to differentiation. The total level of global methylation and the degree of non-CpG methylation is inversely proportional to the level of differentiation.

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Carcinogenesis is a complex multifactorial process of the transformation of normal cells into malignant cells, and is characterized by many biologically significant and interdependent alterations triggered by the mutational and/or non-mutational (i.e., epigenetic) events. One of these events, specific to all types of cancer, is alterations in DNA methylation. Aberrant DNA

According to the mechanism of cancer causation, all carcinogenic agents may be divided into genotoxic (carcinogenic agents that interact with DNA) and non-genotoxic (carcinogenic agents causing tumor by mechanisms other than directly damaging DNA) carcinogens (Figure 2). Exposure to genotoxic carcinogens induces genotoxic and non-genotoxic effects in the DNA methylome, whereas exposure to non-genotoxic carcinogens causes non-genotoxic effects only. Genotoxic effects on the DNA methylome are consist of increased carcinogen–DNAadduct formation at methylated CpG sites and subsequent elevated mutation rates; however, genotoxic effects may cause also non-genotoxic events in the DNA methylome by compro‐ mising ability of the DNA methylation machinery to methylate DNA accurately. Nongenotoxic effects of both genotoxic and non-genotoxic carcinogens consist of global loss of DNA methylation, gene-specific hypermethylation, and gene-specific hypomethylation. Non-

methylation is frequently observed and considered to be a hallmark of cancers.

**5.1. DNA methylome alterations induced by carcinogens**

## *4.3.3. Multipotent neural progenitor cells*

The central nervous system (CNS) is composed of three major cell types-neurons, astrocytes, and oligodendrocytes-which differentiate from common multipotent neural stem cells (NSCs). Comparisons between ESCs, NSCs and terminally differentiated neurons demonstrate that the majority of de novo methylated genes are already present in NSCs, suggesting that the bulk of DNA methylation changes during differentiation is associated with a loss of pluripotency and a commitment to a multipotent state, rather than terminal differentiation [28]. Interest‐ ingly, when NSCs further differentiate into astroglial lineage, selective promoter demethyla‐ tion occurs in glial marker genes, including GFAP and S100B [31, 32]. Loss of methylation using 5-azacytidine (5-azaC) also triggers premature glial differentiation [31]. Consistently, Dnmt1 deficient NSCs precociously differentiate into astroglial cells, which have been linked to increase JAK-STAT signaling and demethylation of the STAT1 and GFAP promoters [32].

### **4.4. DNA methylations in cell reprogramming**

Epigenetic marks can be reset and usually result in the gain of developmental potential, called epigenetic reprogramming. Researches indicated that mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors Oct4, Sox2, Klf4 and c-Myc [33-35]. The ultimate aim of research on cell reprogramming is to create iPSC that is identical to embryonic stem cells (ESC) and differen‐ tiates into tissue specific cell types with intact function, which will pave the way for great advances in regenerative medicine in the future.

### *4.4.1. 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency*

Although 5-hydroxymethylcytosine (5hmC) was discovered several decades ago, it became a major focus of epigenomic research only after it was recently identified in murine brain and stem cell DNA. 5hmC is an oxidative product of 5-methylcytosine (5mC) which catalyzed by the ten eleven translocation (TET) family of enzymes [36]. TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs reprogramming and iPSC-hESC differ‐ ences [37]. Wang et al [37] found that 5hmC levels is increased significantly during reprog‐ ramming to human iPSCs mainly owing to TET1 activation, and this hydroxymethylation change is critical for optimal epigentic reprogramming, but does not compromise primed pluripotency.

#### *4.4.2. Non-CpG cytosine methylation in cells reprogramming*

stem cells [29]. Changes in the DNA methylation profile have a critical role in the divi‐ sion of these stem cells into the myeloid and lymphoid lineages and in the establishment of a specific phenotype and functionality in each terminally differentiated cell type [30]. HSCs deficient in both Dnmt3a and 3b show a loss of proliferative ability but retain differentiation potential, suggesting de novo methylation is important for self-renewal in HSCs [28]. The aberrant DNA methylation has been associated with several immune

The central nervous system (CNS) is composed of three major cell types-neurons, astrocytes, and oligodendrocytes-which differentiate from common multipotent neural stem cells (NSCs). Comparisons between ESCs, NSCs and terminally differentiated neurons demonstrate that the majority of de novo methylated genes are already present in NSCs, suggesting that the bulk of DNA methylation changes during differentiation is associated with a loss of pluripotency and a commitment to a multipotent state, rather than terminal differentiation [28]. Interest‐ ingly, when NSCs further differentiate into astroglial lineage, selective promoter demethyla‐ tion occurs in glial marker genes, including GFAP and S100B [31, 32]. Loss of methylation using 5-azacytidine (5-azaC) also triggers premature glial differentiation [31]. Consistently, Dnmt1 deficient NSCs precociously differentiate into astroglial cells, which have been linked to increase JAK-STAT signaling and demethylation of the STAT1 and GFAP promoters [32].

Epigenetic marks can be reset and usually result in the gain of developmental potential, called epigenetic reprogramming. Researches indicated that mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors Oct4, Sox2, Klf4 and c-Myc [33-35]. The ultimate aim of research on cell reprogramming is to create iPSC that is identical to embryonic stem cells (ESC) and differen‐ tiates into tissue specific cell types with intact function, which will pave the way for great

*4.4.1. 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to*

Although 5-hydroxymethylcytosine (5hmC) was discovered several decades ago, it became a major focus of epigenomic research only after it was recently identified in murine brain and stem cell DNA. 5hmC is an oxidative product of 5-methylcytosine (5mC) which catalyzed by the ten eleven translocation (TET) family of enzymes [36]. TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs reprogramming and iPSC-hESC differ‐ ences [37]. Wang et al [37] found that 5hmC levels is increased significantly during reprog‐ ramming to human iPSCs mainly owing to TET1 activation, and this hydroxymethylation change is critical for optimal epigentic reprogramming, but does not compromise primed

deficiencies and autoimmune disorders [30].

**4.4. DNA methylations in cell reprogramming**

advances in regenerative medicine in the future.

*pluripotency*

pluripotency.

*4.3.3. Multipotent neural progenitor cells*

154 Epigenetics and Epigenomics

Although methylation mainly occurs on the cytosines in the CpG dinucleotide context, non-CG methylation (mCH:DNA methylation targeting CpA, CpT, and CpC dinucleotides) is prevalent in brain, oocytes and pluripotent stem cell [38-40]. Compared to non-growing oocytes (NGOs), germinal vesicle oocytes (GVOs) were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome[38]. Shirane et al [39] found that nearly two-thirds of all meth‐ ylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Ziller et al [40] reported a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types, and confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Non-CpG cytosine methylation has been identified at a high level in stem cells and reprogrammed progenitor cells, indicating that loss of this form of methylation may be critical in the path from pluripotency to differentiation. The total level of global methylation and the degree of non-CpG methylation is inversely proportional to the level of differentiation.

## **5. Methylomes in cancer**

Carcinogenesis is a complex multifactorial process of the transformation of normal cells into malignant cells, and is characterized by many biologically significant and interdependent alterations triggered by the mutational and/or non-mutational (i.e., epigenetic) events. One of these events, specific to all types of cancer, is alterations in DNA methylation. Aberrant DNA methylation is frequently observed and considered to be a hallmark of cancers.

#### **5.1. DNA methylome alterations induced by carcinogens**

According to the mechanism of cancer causation, all carcinogenic agents may be divided into genotoxic (carcinogenic agents that interact with DNA) and non-genotoxic (carcinogenic agents causing tumor by mechanisms other than directly damaging DNA) carcinogens (Figure 2). Exposure to genotoxic carcinogens induces genotoxic and non-genotoxic effects in the DNA methylome, whereas exposure to non-genotoxic carcinogens causes non-genotoxic effects only. Genotoxic effects on the DNA methylome are consist of increased carcinogen–DNAadduct formation at methylated CpG sites and subsequent elevated mutation rates; however, genotoxic effects may cause also non-genotoxic events in the DNA methylome by compro‐ mising ability of the DNA methylation machinery to methylate DNA accurately. Nongenotoxic effects of both genotoxic and non-genotoxic carcinogens consist of global loss of DNA methylation, gene-specific hypermethylation, and gene-specific hypomethylation. Nongenotoxic global DNA hypomethylation leads to genotoxic events such as elevated mutation rates and genome instability. Gene specific DNA hypermethylation of critical tumor suppres‐ sor genes causes transcriptional repression and the loss of gene function. In contrast, gene specific DNA hypomethylation induces activation of oncogenes and tumor-promoting genes. Silencing of DNA repair genes, e.g., MGMT, BRCA1 and MLH1, or activation of xenobiotic metabolizing genes, e.g., CYP1A1, may elevate mutation rates indirectly.

growing evidence for the importance of non-CpG island-promoter methylation in cancer, including methylation of CpG island shores [51], non-CpG promoters [52], and coding regions [53], which results in gene silencing. A mechanistic link between DNA hypermethylation and carcinogenesis is epigenetic silencing of critical tumor-suppressor genes, such as cyclindependent kinase inhibitor 2A (CDKN2A; p16INK4A), secreted frizzled-related protein genes (SFRPs), adenomatous polyposis coli (APC), Ras association (RalGDS/AF-6) domain family member 1 (RASSF1A) [54], et al. It has been established unequivocally that role of epigeneti‐ cally-driven gene silencing has been the main mechanism favoring tumor development and progression. This overshadowed the importance of gene-specific hypomethylation in cancer; however, accumulating evidence indicates that the hypomethylation of "normally" methylat‐ ed CpG island-containing genes also plays a significant role in tumor development. Currently, several hypomethylated tumor-promoting genes, including S100 calcium binding protein A4 (S100A4), plasminogen activator, urokinase (UPA), heparanase (HPA), synuclein, gamma (SNCG), trefoil factor 3 (TFF3), and flap structure-specific endonuclease 1 (FEN1), have been

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Anyway, cancer-linked gene-specific DNA hypermethylation and hypomethylation are associated with the well-established hallmarks of cancer, including the acquisition of persistent proliferative signaling, resistance to cell death, evasion of growth suppression, replicative immortality, inflammation, deregulation of energy metabolism, induction of angiogenesis, and activation of invasion, complement and enhance each other in the disruption of cellular homeostasis favoring cancer development [56]. Next, as examples, the role of DNA methyla‐ tion alterations in carcinogenesis was demonstrated in chronic lymphocytic leukemia and

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia of adults in western countries. Next-generation sequencing of whole genomes, exomes and DNA methylomes in CLL has provided the first comprehensive view of somatic mutations and methylation changes

Two decades ago, high-pressure liquid chromatography (HPLC) analysis revealed the genome DNA of CLL to be globally hypomethylated relative to healthy controls. The subsequent study found that aberrant hypomethylation of repetitive sequences, such as ALU, LINE and SATα leading to genomic instability may be a contributing factor in the increased propensity of TP53 deleted/mutated cases to acquire CLL genomic alterations [57]. Recently, next-generation sequencing of the DNA methylome has also noted gene body hypomethylation to be partic‐ ularly widespread within enhancer regions in CLL patients [57-59]. After the CLL genome was discovered to be hypomethylated, hypomethylation of oncogene is found to correlate with increased protein expression in CLL. Such as, BCL2, a key anti-apoptotic gene, MDR1, the multiple drug resistance gene, and TCL1, an activator of NF-κB, were subsequently found to

in this disease. Here, we mainly elaborate the change of DNA methylomes in CLL.

*5.2.1. Hypomethylation contributes to the genomic instability and oncogene activation in CLL*

identified in major human cancers [55].

**5.2. Methylome in chronic lymphocytic leukemia**

be hypomethylated and upregulated in CLL [57].

glioma.

**Figure 2.** Alterations of the DNA methylome induced by carcinogens

#### *5.1.1. Global DNA hypomethylation in cancer*

DNA hypomethylation arises mainly from the loss of methylation at normally heavily methylated areas of genome. The loss of global DNA methylation is one of the most common DNA methylome alterations in human cancers, which is closely related to carcinogenesis. First, genomic demethylation causes a significant elevation in mutation rates] and aberrant activa‐ tion of"normally"silenced tumor promoting genes [41-43]. Second, hypomethylation of DNA results in the loss of genomic imprinting (LOI), which is currently considered as one of the earliest and most frequent alterations in human tumors [44-46]. Third, demethylation of repetitive sequences, such as long interspersed nucleotide elements (LINE)-1, short inter‐ spersed nucleotide elements (SINE), and retroviral intracisternal. Alu elements may cause chromosomal abnormalities and genomic instability via the induction of permissive transcrip‐ tional activity of repetitive elements [47-48]. Finally, the recent research indicated that the loss of 5hmC has been found in a broad spectrum of solid tumors, including lung, breast, brain, gastric, and colorectal cancers [49-50].

#### *5.1.2. Cancer-linked gene-specific DNA hypermethylation or hypomethylation*

DNA hypermethylation is the most extensively studied epigenetic abnormality in cancer, and the hypermethylation of promoter CpG islands causes permanent and stable transcriptional silencing of a wide range of protein-coding genes and non-coding RNA genes. There is also growing evidence for the importance of non-CpG island-promoter methylation in cancer, including methylation of CpG island shores [51], non-CpG promoters [52], and coding regions [53], which results in gene silencing. A mechanistic link between DNA hypermethylation and carcinogenesis is epigenetic silencing of critical tumor-suppressor genes, such as cyclindependent kinase inhibitor 2A (CDKN2A; p16INK4A), secreted frizzled-related protein genes (SFRPs), adenomatous polyposis coli (APC), Ras association (RalGDS/AF-6) domain family member 1 (RASSF1A) [54], et al. It has been established unequivocally that role of epigeneti‐ cally-driven gene silencing has been the main mechanism favoring tumor development and progression. This overshadowed the importance of gene-specific hypomethylation in cancer; however, accumulating evidence indicates that the hypomethylation of "normally" methylat‐ ed CpG island-containing genes also plays a significant role in tumor development. Currently, several hypomethylated tumor-promoting genes, including S100 calcium binding protein A4 (S100A4), plasminogen activator, urokinase (UPA), heparanase (HPA), synuclein, gamma (SNCG), trefoil factor 3 (TFF3), and flap structure-specific endonuclease 1 (FEN1), have been identified in major human cancers [55].

Anyway, cancer-linked gene-specific DNA hypermethylation and hypomethylation are associated with the well-established hallmarks of cancer, including the acquisition of persistent proliferative signaling, resistance to cell death, evasion of growth suppression, replicative immortality, inflammation, deregulation of energy metabolism, induction of angiogenesis, and activation of invasion, complement and enhance each other in the disruption of cellular homeostasis favoring cancer development [56]. Next, as examples, the role of DNA methyla‐ tion alterations in carcinogenesis was demonstrated in chronic lymphocytic leukemia and glioma.

### **5.2. Methylome in chronic lymphocytic leukemia**

genotoxic global DNA hypomethylation leads to genotoxic events such as elevated mutation rates and genome instability. Gene specific DNA hypermethylation of critical tumor suppres‐ sor genes causes transcriptional repression and the loss of gene function. In contrast, gene specific DNA hypomethylation induces activation of oncogenes and tumor-promoting genes. Silencing of DNA repair genes, e.g., MGMT, BRCA1 and MLH1, or activation of xenobiotic

DNA hypomethylation arises mainly from the loss of methylation at normally heavily methylated areas of genome. The loss of global DNA methylation is one of the most common DNA methylome alterations in human cancers, which is closely related to carcinogenesis. First, genomic demethylation causes a significant elevation in mutation rates] and aberrant activa‐ tion of"normally"silenced tumor promoting genes [41-43]. Second, hypomethylation of DNA results in the loss of genomic imprinting (LOI), which is currently considered as one of the earliest and most frequent alterations in human tumors [44-46]. Third, demethylation of repetitive sequences, such as long interspersed nucleotide elements (LINE)-1, short inter‐ spersed nucleotide elements (SINE), and retroviral intracisternal. Alu elements may cause chromosomal abnormalities and genomic instability via the induction of permissive transcrip‐ tional activity of repetitive elements [47-48]. Finally, the recent research indicated that the loss of 5hmC has been found in a broad spectrum of solid tumors, including lung, breast, brain,

DNA hypermethylation is the most extensively studied epigenetic abnormality in cancer, and the hypermethylation of promoter CpG islands causes permanent and stable transcriptional silencing of a wide range of protein-coding genes and non-coding RNA genes. There is also

metabolizing genes, e.g., CYP1A1, may elevate mutation rates indirectly.

**Figure 2.** Alterations of the DNA methylome induced by carcinogens

*5.1.1. Global DNA hypomethylation in cancer*

156 Epigenetics and Epigenomics

gastric, and colorectal cancers [49-50].

*5.1.2. Cancer-linked gene-specific DNA hypermethylation or hypomethylation*

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia of adults in western countries. Next-generation sequencing of whole genomes, exomes and DNA methylomes in CLL has provided the first comprehensive view of somatic mutations and methylation changes in this disease. Here, we mainly elaborate the change of DNA methylomes in CLL.

#### *5.2.1. Hypomethylation contributes to the genomic instability and oncogene activation in CLL*

Two decades ago, high-pressure liquid chromatography (HPLC) analysis revealed the genome DNA of CLL to be globally hypomethylated relative to healthy controls. The subsequent study found that aberrant hypomethylation of repetitive sequences, such as ALU, LINE and SATα leading to genomic instability may be a contributing factor in the increased propensity of TP53 deleted/mutated cases to acquire CLL genomic alterations [57]. Recently, next-generation sequencing of the DNA methylome has also noted gene body hypomethylation to be partic‐ ularly widespread within enhancer regions in CLL patients [57-59]. After the CLL genome was discovered to be hypomethylated, hypomethylation of oncogene is found to correlate with increased protein expression in CLL. Such as, BCL2, a key anti-apoptotic gene, MDR1, the multiple drug resistance gene, and TCL1, an activator of NF-κB, were subsequently found to be hypomethylated and upregulated in CLL [57].

## *5.2.2. Cells origin and subtypes of CLL on basis of genome and methylome*

Because of the differences in the immunoglobulin heavy variation (IGHV) mutational status and B-cell receptor reactivity, chronic lymphocytic leukemia was classified into two subtypes: chronic lymphocytic leukemia lacking significant somatic IGHV mutation (uCLL) and chronic lymphocytic leukemia with significant somatic IGHV mutation (mCLL), which was derived from naive B cells and memory B cells [60]. Most mutated genes cluster in a few molecular pathways that are also differentially represented in the two subtypes of CLL. Mutations in NOTCH1 signaling, mRNA splicing, processing and transport, and DNA damage response pathways are more common in uCLL, whereas mutations in the innate inflammatory pathway occur predominantly in mCLL.

gene expression and involved in tumor development, we focused on the analysis of DMRs in glioma which were mapped to gene promoter regions. The 216 promoter hypermethylated genes and 60 promoter hypomethylated genes identified by MeDIP-chip were analyzed according to their chromosomal location and the physical distribution of these loci was further analyzed (Fig 3D). Except that there were intensive promoter hypermethylation genes in 1, 2, 3, 17 and X chromosomes, the promoter hypermethylated genes were found to be distributed evenly in other chromosomes. While promoter hypomethylated genes mainly distributed in 1, 11, 16, 19, 20 and 22 chromosomes, the number of genes in these chromosomes account for

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**Figure 3.** Genome-wide analysis of DMRs in primary glioma. (A) Number of differentially methylated regions that are associated with or without genes. (B) Distribution of diifferentially methylated regions associated with genes. Most of the identified DMRs associated with genes were mapped to gene promoters. (C) Number of DMRs which were both gene promoters and CpG islands. (D) Chromosomal distribution of 216 promoter hypermethylated genes and 60 pro‐

**6. The connected networks of DNA methylation, histone modifications,**

Epigenetic mechanisms include: DNA methylation; histone tail modifications; chromatin remodeling; and noncoding RNA expression. DNA methylation is essential for a properly functioning genome through its roles in the maintenance of chromatin structure, chromosome stability and transcription. Histones are the protein moiety around which DNA is packaged within the chromatin, and they can suffer a variety of post-translational modifications of their

the majority of the total promoter hypomethylated genes [61].

moter hypomethylated genes.

**transcript factor and miRNAs**

B cells at different maturation stages require a different level of DNA methylation reprogram‐ ming to give rise to uCLL and mCLL. Microarray analysis of a large series of patients indicates that uCLLs acquire approximately seven times more DNA methylation changes than mCLL compared with their respective cells of origin. In particular, two-thirds of the DNA methylation changes that take place in the transformation of naive B cells into uCLL can also be detected in their physiological differentiation into memory B cells [58].

By genome-wide differential DNA methylation profile analysis in uCLL and mCLL, CLL was derived from three different B-cell subpopulations: uCLL resembles both native B cells (IgD + and CD27- ) and CD5+ pregerminal center mature B cells (CD5+ , IgD+ , and CD27- ), whereas mCLL is more similar to non class-switched and class-switched memory B cells (IgM/ D+ or IgA/G+ , CD27+ ). The third group of CLL was accompied with an intermediate DNA methyla‐ tion pattern and enriched for mCLLs with a significantly lower level of somatic IGHV mutations [58]. This group might be derived from a third B-cell type, for example, an antigenexperienced, germinal center-independent B cell that has acquired low levels of somatic hypermutations [60].

#### **5.3. Methylomes in glioma**

Glioma is the most frequent and devastating primary brain tumor in adults. Aberrant DNA methylation contributes to glioma development and progression.We employed MeDIP-chip to investigate the whole-genome differential methylation patterns between glioma and normal brain samples. We identified 524 hypermethylated and 114 hypomethylated differential regions in the primary gliomas. Intriguingly, some of the human genome differential methyl‐ ation regions (DMRs), 199 hypermethylation and 30 hypomethylated differential regions were mapped to genomic regions without any gene annotation (Fig 3A). Only 325 hypermethylation and 74 hypomethylation differential regions were mapped to annotated genes regions, including promoter, intragenic and downstream of genes (Fig3A). A great percentage of DMRs, 63.0% (216) of hypermethylated and 79.0% (60) of hypomethylated differential regions, was mapped to promoter regions of known genes (Fig 3B). 53 hypermethylated and 27 hypomethylated differential regions were mapped to the regions which were both promoter of known genes and CpG islands (Fig 3C). Thus, we identified many novel DMRs that reside in promoters, intragenic, downstream of known genes and unannotated genomic regions in primary gliomas. Since change of promoter methylation status may have close related with gene expression and involved in tumor development, we focused on the analysis of DMRs in glioma which were mapped to gene promoter regions. The 216 promoter hypermethylated genes and 60 promoter hypomethylated genes identified by MeDIP-chip were analyzed according to their chromosomal location and the physical distribution of these loci was further analyzed (Fig 3D). Except that there were intensive promoter hypermethylation genes in 1, 2, 3, 17 and X chromosomes, the promoter hypermethylated genes were found to be distributed evenly in other chromosomes. While promoter hypomethylated genes mainly distributed in 1, 11, 16, 19, 20 and 22 chromosomes, the number of genes in these chromosomes account for the majority of the total promoter hypomethylated genes [61].

*5.2.2. Cells origin and subtypes of CLL on basis of genome and methylome*

in their physiological differentiation into memory B cells [58].

occur predominantly in mCLL.

158 Epigenetics and Epigenomics

) and CD5+

+

and CD27-

, CD27+

hypermutations [60].

**5.3. Methylomes in glioma**

IgA/G+

Because of the differences in the immunoglobulin heavy variation (IGHV) mutational status and B-cell receptor reactivity, chronic lymphocytic leukemia was classified into two subtypes: chronic lymphocytic leukemia lacking significant somatic IGHV mutation (uCLL) and chronic lymphocytic leukemia with significant somatic IGHV mutation (mCLL), which was derived from naive B cells and memory B cells [60]. Most mutated genes cluster in a few molecular pathways that are also differentially represented in the two subtypes of CLL. Mutations in NOTCH1 signaling, mRNA splicing, processing and transport, and DNA damage response pathways are more common in uCLL, whereas mutations in the innate inflammatory pathway

B cells at different maturation stages require a different level of DNA methylation reprogram‐ ming to give rise to uCLL and mCLL. Microarray analysis of a large series of patients indicates that uCLLs acquire approximately seven times more DNA methylation changes than mCLL compared with their respective cells of origin. In particular, two-thirds of the DNA methylation changes that take place in the transformation of naive B cells into uCLL can also be detected

By genome-wide differential DNA methylation profile analysis in uCLL and mCLL, CLL was derived from three different B-cell subpopulations: uCLL resembles both native B cells (IgD

mCLL is more similar to non class-switched and class-switched memory B cells (IgM/ D+ or

tion pattern and enriched for mCLLs with a significantly lower level of somatic IGHV mutations [58]. This group might be derived from a third B-cell type, for example, an antigenexperienced, germinal center-independent B cell that has acquired low levels of somatic

Glioma is the most frequent and devastating primary brain tumor in adults. Aberrant DNA methylation contributes to glioma development and progression.We employed MeDIP-chip to investigate the whole-genome differential methylation patterns between glioma and normal brain samples. We identified 524 hypermethylated and 114 hypomethylated differential regions in the primary gliomas. Intriguingly, some of the human genome differential methyl‐ ation regions (DMRs), 199 hypermethylation and 30 hypomethylated differential regions were mapped to genomic regions without any gene annotation (Fig 3A). Only 325 hypermethylation and 74 hypomethylation differential regions were mapped to annotated genes regions, including promoter, intragenic and downstream of genes (Fig3A). A great percentage of DMRs, 63.0% (216) of hypermethylated and 79.0% (60) of hypomethylated differential regions, was mapped to promoter regions of known genes (Fig 3B). 53 hypermethylated and 27 hypomethylated differential regions were mapped to the regions which were both promoter of known genes and CpG islands (Fig 3C). Thus, we identified many novel DMRs that reside in promoters, intragenic, downstream of known genes and unannotated genomic regions in primary gliomas. Since change of promoter methylation status may have close related with

). The third group of CLL was accompied with an intermediate DNA methyla‐

, IgD+

, and CD27-

), whereas

pregerminal center mature B cells (CD5+

**Figure 3.** Genome-wide analysis of DMRs in primary glioma. (A) Number of differentially methylated regions that are associated with or without genes. (B) Distribution of diifferentially methylated regions associated with genes. Most of the identified DMRs associated with genes were mapped to gene promoters. (C) Number of DMRs which were both gene promoters and CpG islands. (D) Chromosomal distribution of 216 promoter hypermethylated genes and 60 pro‐ moter hypomethylated genes.

## **6. The connected networks of DNA methylation, histone modifications, transcript factor and miRNAs**

Epigenetic mechanisms include: DNA methylation; histone tail modifications; chromatin remodeling; and noncoding RNA expression. DNA methylation is essential for a properly functioning genome through its roles in the maintenance of chromatin structure, chromosome stability and transcription. Histones are the protein moiety around which DNA is packaged within the chromatin, and they can suffer a variety of post-translational modifications of their N-terminal tails, including acetylation, methylation, phosphorylation, sumoylation, ubiquiti‐ nation and ADP ribosylation [62,63]. miRNAs are ~20–22 nucleotide non-coding RNA molecules that tend to negatively regulate genes by binding to the 3' untranslated region of the target mRNA via the RNA-induced silencing complex causing mRNA destabilization and/ or translational inhibition [64,65]. Growing evidence supports a role for miRNAs as both targets and effectors in aberrant mechanisms of DNA methylation [66,67]. Meanwhile, miRNAs are also invovlved in the control of DNA methylation by targeting the DNA meth‐ ylation machinery [68, 69]. In this section, we combined with our own work to demonstrate the connected networks of DNA methylation, histone modifications, transcript factor and miRNAs in glioma. On the basis DNA methylome of glioma, we identified fifteen new methylated genes including 9 hypermethylated genes(ANKDD1A, GAD1, SIX3, SST, PHOX2B, PCDHA8,PCDHA13, HIST1H3E and LRRC4) and 6 hypomethylated genes (F10, POTEH, CPEB1, LMO3, ELFN2 and PRDM16) were validated by the Sequenom MassARRAY platform and bisulfite sequencing (BSP) in glioma. Aberrant promoter methylation and changed histone modifications were associated with gene abnormal expression in glioma. miR-185 targets the DNA methyltransferases 1 and regulates global DNA methylation [61], however, miR-101 regulates histone methylation modication of hypomethylated gene CPEB1 by targeting EZH2 and EED, and DNMT3A, and affected their methylation level and expres‐ sion in glioma [70].

Transcription of miR-182 was induced by transcription factor AP-2 predicted by online softwares and confimed by ChIP. According to our previous results, miR-182 was verified to inhibit the expression of LRRC4, and LRRC4 might inhibit the expression and transcription of AP-2 through negatively regulating the ERK/MAPK and PI-3K/AKT signaling pathways. It's indicated that the LRRC4-AP-2-miR-182-LRRC4 loop formed among LRRC4, miR-182 and

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http://dx.doi.org/10.5772/57535

miR-185 could function as a tumor suppressor gene. It's certified that miR-185 could inhibit glioma cell growth, motility and invasion identified by MTT, scratch test and transwell test [78-80]. DNMT1 is one of the most important DNA methyltransferase which maintains methylation. Our research showed that overexpression of miR-185 could inhibit DNMT1 and reduce global methylation by HPLC-DAD, and decreased the expression of nine new hyper‐ methylated genes (LRRC4, ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3 and SST) [61]. Hence, miR-185 was considered to inhibit glioma cells growth and migration by targeting DNMT1, reducing global methylation and recovering expression of

miR-185 also was predicted to participate in Rho GTPase activity based on GO analysis, while CDC42 and RhoA were the main elements regulating Rho GTPase. Then CDC42 and RhoA were identified to be the direct targets of miR-185[61]. Further, CDC42 and RhoA were inversely correlated with miR-185 expression in gliomas. miR-185 was clarified to mediate glioma cell growth and migration by inhibiting CDC42 and RhoA and VEGFA indirectly [61].

It's verified that overexpressing LRRC4 could increase the expression of miR-185, while miR-185 could regulate global methylation by inhibiting DNA methyltranferase DNMT1 and increasing the expression of such hypermethylation gene as LRRC4. There may be form LRRC4-miR-185-DNMT1-LRRC4 loop which LRRC4 are to be as core the loop, miR185 and DNMT1 that participating in glioma development. In addition, DNMT1 was positively regulated by SP1, and it could increase the expression of LRRC4, while LRRC4 could also inhibit SP1 by negatively regulate ERK/MAPK and PI-3K/AKT signal pathway. So that the LRRC4-SP1-DNMT1-LRRC4 loop formed among LRRC4, SP1 and DNMT1 took part in the

In conclusion, development of glioma is the pathological processing with multiple genes and multi-stages. Genes, miRNAs and DNA methylation play an important role in glioma formation. They may support or antagonize each other and construct complicated network in glioma. In sum of above study, at the time of LRRC4 regulating miRNAs as a tumor suppressor, those miRNAs regulated by LRRC4 were found to regulate the binding of transcription factors to DNA in their targets mediated signaling pathways by directly targeting genes (such as LRRC4), or regulate methylation and expression of such hypermethylation genes as LRRC4 by directly targeting DNA methyltransferase and controlling global methylation. And multipases regulation loops which the core was LRRC4 were formed. They were LRRC4-AP-2 miR-182-LRRC4, LRRC4-miR-185-DNMT1-LRRC4 and LRRC4-SP1-DNMT1-LRRC4. These

*6.1.2. LRRC4-miR-185/SP1-DNMT1-LRRC4 loop played an important role in glioma*

AP-2 was involved in glioma development [77].

such hypermethylation genes as LRRC4.

glioma formation.

#### **6.1. The regulation networks between hypermethylated gene, miRNA, transcript factor and target gene in glioma**

Leucine-rich repeat C4 (LRRC4) gene, a new hypermethylated gene identified by DNA methylome of glioma, are highly specific to brain tissue and it behaved as a tumor suppressor gene in the pathogenesis of gliomas. Methylation of the LRRC4 promoter has been considered as one of the important mechanisms inactivating LRRC4 in gliomas. Exogenous overexpres‐ sion of LRRC4 could inhibit glioma cells growth and arrest glioma cells in the G0/G1 phase of the cell cycle. Induction of LRRC4 expression inhibited glioma cell proliferation and invasion by downregulating the ERK/MAPK and PI-3K/AKT signaling pathways [71-75].

#### *6.1.1. LRRC4-AP-2-miR-182-LRRC4 loop played important role in the pathogenesis of glioma*

It's known that miRNAs took part in proliferation and growth in glioma cells. miR-182 or miR-381 overexpression could promote glioma cell growth in vivo and in vitro. Therefore, they were considered to be potential therapeutic biomarker in glioma. miR-182 or miR-381 silencing could arrest glioma cells in the G0/G1 phase of the cell cycle and inhibit glioma cells growth by upregulating phosphorylated Rb and suppressing E2F3. LRRC4 was the co-target gene of miR-182 and miR-381. The expression of miR-182, miR-381 and BRD7 were inversely correlated with LRRC4 expression in gliomas. miR-182 and miR-381 silencing was found to inhibit the expression of BRD7, upregulate phosphorylated Rb, suppress E2F3, arrest glioma cells in the G0/G1 phase of the cell cycle, inhibit glioma cells growth and induce differentiation of glioma cells to astrocyte-like cell by upregulating LRRC4 and suppressing LRRC4-mediated binding of AP-2/SP1/E2F6/c-Myc to BRD7 in ERK/MAPK and PI-3K/AKT signal pathways [76].

Transcription of miR-182 was induced by transcription factor AP-2 predicted by online softwares and confimed by ChIP. According to our previous results, miR-182 was verified to inhibit the expression of LRRC4, and LRRC4 might inhibit the expression and transcription of AP-2 through negatively regulating the ERK/MAPK and PI-3K/AKT signaling pathways. It's indicated that the LRRC4-AP-2-miR-182-LRRC4 loop formed among LRRC4, miR-182 and AP-2 was involved in glioma development [77].

### *6.1.2. LRRC4-miR-185/SP1-DNMT1-LRRC4 loop played an important role in glioma*

N-terminal tails, including acetylation, methylation, phosphorylation, sumoylation, ubiquiti‐ nation and ADP ribosylation [62,63]. miRNAs are ~20–22 nucleotide non-coding RNA molecules that tend to negatively regulate genes by binding to the 3' untranslated region of the target mRNA via the RNA-induced silencing complex causing mRNA destabilization and/ or translational inhibition [64,65]. Growing evidence supports a role for miRNAs as both targets and effectors in aberrant mechanisms of DNA methylation [66,67]. Meanwhile, miRNAs are also invovlved in the control of DNA methylation by targeting the DNA meth‐ ylation machinery [68, 69]. In this section, we combined with our own work to demonstrate the connected networks of DNA methylation, histone modifications, transcript factor and miRNAs in glioma. On the basis DNA methylome of glioma, we identified fifteen new methylated genes including 9 hypermethylated genes(ANKDD1A, GAD1, SIX3, SST, PHOX2B, PCDHA8,PCDHA13, HIST1H3E and LRRC4) and 6 hypomethylated genes (F10, POTEH, CPEB1, LMO3, ELFN2 and PRDM16) were validated by the Sequenom MassARRAY platform and bisulfite sequencing (BSP) in glioma. Aberrant promoter methylation and changed histone modifications were associated with gene abnormal expression in glioma. miR-185 targets the DNA methyltransferases 1 and regulates global DNA methylation [61], however, miR-101 regulates histone methylation modication of hypomethylated gene CPEB1 by targeting EZH2 and EED, and DNMT3A, and affected their methylation level and expres‐

**6.1. The regulation networks between hypermethylated gene, miRNA, transcript factor and**

Leucine-rich repeat C4 (LRRC4) gene, a new hypermethylated gene identified by DNA methylome of glioma, are highly specific to brain tissue and it behaved as a tumor suppressor gene in the pathogenesis of gliomas. Methylation of the LRRC4 promoter has been considered as one of the important mechanisms inactivating LRRC4 in gliomas. Exogenous overexpres‐ sion of LRRC4 could inhibit glioma cells growth and arrest glioma cells in the G0/G1 phase of the cell cycle. Induction of LRRC4 expression inhibited glioma cell proliferation and invasion

by downregulating the ERK/MAPK and PI-3K/AKT signaling pathways [71-75].

*6.1.1. LRRC4-AP-2-miR-182-LRRC4 loop played important role in the pathogenesis of glioma*

It's known that miRNAs took part in proliferation and growth in glioma cells. miR-182 or miR-381 overexpression could promote glioma cell growth in vivo and in vitro. Therefore, they were considered to be potential therapeutic biomarker in glioma. miR-182 or miR-381 silencing could arrest glioma cells in the G0/G1 phase of the cell cycle and inhibit glioma cells growth by upregulating phosphorylated Rb and suppressing E2F3. LRRC4 was the co-target gene of miR-182 and miR-381. The expression of miR-182, miR-381 and BRD7 were inversely correlated with LRRC4 expression in gliomas. miR-182 and miR-381 silencing was found to inhibit the expression of BRD7, upregulate phosphorylated Rb, suppress E2F3, arrest glioma cells in the G0/G1 phase of the cell cycle, inhibit glioma cells growth and induce differentiation of glioma cells to astrocyte-like cell by upregulating LRRC4 and suppressing LRRC4-mediated binding of AP-2/SP1/E2F6/c-Myc to BRD7 in ERK/MAPK and PI-3K/AKT signal pathways [76].

sion in glioma [70].

160 Epigenetics and Epigenomics

**target gene in glioma**

miR-185 could function as a tumor suppressor gene. It's certified that miR-185 could inhibit glioma cell growth, motility and invasion identified by MTT, scratch test and transwell test [78-80]. DNMT1 is one of the most important DNA methyltransferase which maintains methylation. Our research showed that overexpression of miR-185 could inhibit DNMT1 and reduce global methylation by HPLC-DAD, and decreased the expression of nine new hyper‐ methylated genes (LRRC4, ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3 and SST) [61]. Hence, miR-185 was considered to inhibit glioma cells growth and migration by targeting DNMT1, reducing global methylation and recovering expression of such hypermethylation genes as LRRC4.

miR-185 also was predicted to participate in Rho GTPase activity based on GO analysis, while CDC42 and RhoA were the main elements regulating Rho GTPase. Then CDC42 and RhoA were identified to be the direct targets of miR-185[61]. Further, CDC42 and RhoA were inversely correlated with miR-185 expression in gliomas. miR-185 was clarified to mediate glioma cell growth and migration by inhibiting CDC42 and RhoA and VEGFA indirectly [61].

It's verified that overexpressing LRRC4 could increase the expression of miR-185, while miR-185 could regulate global methylation by inhibiting DNA methyltranferase DNMT1 and increasing the expression of such hypermethylation gene as LRRC4. There may be form LRRC4-miR-185-DNMT1-LRRC4 loop which LRRC4 are to be as core the loop, miR185 and DNMT1 that participating in glioma development. In addition, DNMT1 was positively regulated by SP1, and it could increase the expression of LRRC4, while LRRC4 could also inhibit SP1 by negatively regulate ERK/MAPK and PI-3K/AKT signal pathway. So that the LRRC4-SP1-DNMT1-LRRC4 loop formed among LRRC4, SP1 and DNMT1 took part in the glioma formation.

In conclusion, development of glioma is the pathological processing with multiple genes and multi-stages. Genes, miRNAs and DNA methylation play an important role in glioma formation. They may support or antagonize each other and construct complicated network in glioma. In sum of above study, at the time of LRRC4 regulating miRNAs as a tumor suppressor, those miRNAs regulated by LRRC4 were found to regulate the binding of transcription factors to DNA in their targets mediated signaling pathways by directly targeting genes (such as LRRC4), or regulate methylation and expression of such hypermethylation genes as LRRC4 by directly targeting DNA methyltransferase and controlling global methylation. And multipases regulation loops which the core was LRRC4 were formed. They were LRRC4-AP-2 miR-182-LRRC4, LRRC4-miR-185-DNMT1-LRRC4 and LRRC4-SP1-DNMT1-LRRC4. These loops participated in glioma development with multiple positive feedback formation among them (Fig 4).

EZH2, EED and DNMT3A, then it recovered the methylation levels of CPEB1 gene promoter,

Methylomes

163

http://dx.doi.org/10.5772/57535

LRRC4 has been a glioma suppressor gene and its hypermethylation and down-expres‐ sion is common in glioma. In order to clarify the mechanism of LRRC4 regulation, miRNAs regulating LRRC4 were predicted. miR-101 was predicted to target LRRC4. Here, we indicated that miR-101 could not bind to 3'UTR of LRRC4, but it remain to upregulate the expression of LRRC4 in glioma cells. miR-101 decreased the occupancy of H3K27me3 at LRRC4 core promoter and induced hypomethylation of LRRC4 by targeting EZH2, EED

Token together, deregulation of gene methylation including hypermethylation and hypome‐ thylation plays an important role in carcinogenesis of glioma. Hypermethylation or hypome‐ thylation of genes and their deregulated expression could be applied to predict the early diagnosis and prognosis of glioma. miRNAs are small noncoding RNA, around 22-24 nucleo‐ tides in size. They could not only directly regulate expression hyper/hypo-methylation genes by binding to 3'-UTR of genes, but also regulate the methylation level and gene expression through histone and DNA methylation modification by targeting histone and DNA methyl‐

**Figure 5.** The regulation networks of miRNA, genes methylation and histone protein modification in glioma

and indirectly down-regulating the expression of these hypomethylation genes.

*H3K27me3 occupancy and hypomethylation level of LRRC4 in glioma cells*

and DNMT3A [81].

transferases (Fig 5).

*6.2.2. miR-101 recovered the expression of hypermethylation gene LRRC4 by down-regulating*

**Figure 4.** The regulation networks of hypermethylated genes, miRNA, DNMT, transcript factors and targets genes in glioma

## **6.2. miR-101 regulates the expression of hypomethylated/hpermethylated genes by different histone protein methylaiton modification**

It's well-known that miRNAs play significant role by regulating gene expression in tumors. We assumed to analyze the upregulation mechanism of hypomethylation genes in the extent of gene regulation by miRNA. Subsequently, we predicted miRNAs which could regulate hypomethylation genes CPEB1. Interestedly, CPEB1was predicted to be a target of miR-101 by online software Targetscan6.0. It's confirmed that miR-101 could bind to the 3'UTR of CPEB1 and inhibit their expression [70].

### *6.2.1. miR-101 indirectly suppressed expression of CPEB1 and affected their methylation levels by targeting EZH2, EED and DNMT3A and regulating histone methylation in glioma cells*

As miR-101 regulated the methylation status and expression of gene through histone modifi‐ cation, it may regulate the methylation status of CPEB1 in the same way. Hence, the effect of miR-101, EZH2 siRNA, EED siRNA and DNMT3A siRNA on histone methylation and expression of CPEB1 was detected. ChIP combining with qRT-PCR and BSP was used to verify that miR-101 decreased the H3K4me2 and H3K27me3 occupancy at CPEB1 core promoter and increased the H3K9me3 and H4K20me3 occupancy at CPEB1 core promoter by targeting EZH2, EED and DNMT3A, then it recovered the methylation levels of CPEB1 gene promoter, and indirectly down-regulating the expression of these hypomethylation genes.

### *6.2.2. miR-101 recovered the expression of hypermethylation gene LRRC4 by down-regulating H3K27me3 occupancy and hypomethylation level of LRRC4 in glioma cells*

loops participated in glioma development with multiple positive feedback formation among

**Figure 4.** The regulation networks of hypermethylated genes, miRNA, DNMT, transcript factors and targets genes in

It's well-known that miRNAs play significant role by regulating gene expression in tumors. We assumed to analyze the upregulation mechanism of hypomethylation genes in the extent of gene regulation by miRNA. Subsequently, we predicted miRNAs which could regulate hypomethylation genes CPEB1. Interestedly, CPEB1was predicted to be a target of miR-101 by online software Targetscan6.0. It's confirmed that miR-101 could bind to the 3'UTR of

*6.2.1. miR-101 indirectly suppressed expression of CPEB1 and affected their methylation levels by*

As miR-101 regulated the methylation status and expression of gene through histone modifi‐ cation, it may regulate the methylation status of CPEB1 in the same way. Hence, the effect of miR-101, EZH2 siRNA, EED siRNA and DNMT3A siRNA on histone methylation and expression of CPEB1 was detected. ChIP combining with qRT-PCR and BSP was used to verify that miR-101 decreased the H3K4me2 and H3K27me3 occupancy at CPEB1 core promoter and increased the H3K9me3 and H4K20me3 occupancy at CPEB1 core promoter by targeting

*targeting EZH2, EED and DNMT3A and regulating histone methylation in glioma cells*

**6.2. miR-101 regulates the expression of hypomethylated/hpermethylated genes by**

**different histone protein methylaiton modification**

CPEB1 and inhibit their expression [70].

them (Fig 4).

162 Epigenetics and Epigenomics

glioma

LRRC4 has been a glioma suppressor gene and its hypermethylation and down-expres‐ sion is common in glioma. In order to clarify the mechanism of LRRC4 regulation, miRNAs regulating LRRC4 were predicted. miR-101 was predicted to target LRRC4. Here, we indicated that miR-101 could not bind to 3'UTR of LRRC4, but it remain to upregulate the expression of LRRC4 in glioma cells. miR-101 decreased the occupancy of H3K27me3 at LRRC4 core promoter and induced hypomethylation of LRRC4 by targeting EZH2, EED and DNMT3A [81].

Token together, deregulation of gene methylation including hypermethylation and hypome‐ thylation plays an important role in carcinogenesis of glioma. Hypermethylation or hypome‐ thylation of genes and their deregulated expression could be applied to predict the early diagnosis and prognosis of glioma. miRNAs are small noncoding RNA, around 22-24 nucleo‐ tides in size. They could not only directly regulate expression hyper/hypo-methylation genes by binding to 3'-UTR of genes, but also regulate the methylation level and gene expression through histone and DNA methylation modification by targeting histone and DNA methyl‐ transferases (Fig 5).

**Figure 5.** The regulation networks of miRNA, genes methylation and histone protein modification in glioma

## **7. Conclusion**

The advances in next-generation sequencing technologies have allowed for mapping of DNA methylation and its derivatives: 5hmC and 5fC at base-pair resolution. These studies have provided key new insights into the function, dynamics and distribution of DNA methylation in vertebrate genomes. In the near future, studies of DM sites and focal DMRs will aid the discovery of transcription factors and transcription regulatory elements involved in control‐ ling the expression of specific genes in vivo. More experiments in model systems will be done to directly test the functionality of DMRs or individual DM sites identified in epigenomic profiles. It is likely that intragenic and distant intergenic changes in DNA methylation will be studied much more than at present for their contribution to diseases involving epigenetic deregulation, especially cancer, immunological diseases and neurological diseases.

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## **Acknowledgements**

This work was supported by grants from National Natural Science Foundation of China (81272297; 81171932) and Hunan Province Natural Sciences Foundations (11JJ1013); Research Fund for the Doctoral Program of Higher Education of China (20110162110037). There are no financial or other relationships that might lead to a conflict of interest.

## **Author details**

Minghua Wu1,2,3\*

Address all correspondence to: wuminghua554@aliyun.com

1 Cancer Research Institute, Central South University, Changsha, Hunan, China

2 Disease genome Research Center, Central South University, Changsha, Hunan, China

3 Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education; Key Labo‐ ratory of Carcinogenesis, Ministry of Health; The Center for Skull Base Surgery and Neuro‐ oncology, Hunan Province, China

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**7. Conclusion**

164 Epigenetics and Epigenomics

**Acknowledgements**

**Author details**

Minghua Wu1,2,3\*

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The advances in next-generation sequencing technologies have allowed for mapping of DNA methylation and its derivatives: 5hmC and 5fC at base-pair resolution. These studies have provided key new insights into the function, dynamics and distribution of DNA methylation in vertebrate genomes. In the near future, studies of DM sites and focal DMRs will aid the discovery of transcription factors and transcription regulatory elements involved in control‐ ling the expression of specific genes in vivo. More experiments in model systems will be done to directly test the functionality of DMRs or individual DM sites identified in epigenomic profiles. It is likely that intragenic and distant intergenic changes in DNA methylation will be studied much more than at present for their contribution to diseases involving epigenetic

deregulation, especially cancer, immunological diseases and neurological diseases.

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1 Cancer Research Institute, Central South University, Changsha, Hunan, China

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2 Disease genome Research Center, Central South University, Changsha, Hunan, China

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Address all correspondence to: wuminghua554@aliyun.com

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**Chapter 7**

**Epigenome, Cancer Prevention and Flavonoids and**

Epigenome is a common name for heritable chemical modifications of DNA and histone molecules, of which DNA methylation and histone acetylation and methylation represent the most studied parts. Nucleosomes, the chromatin building units, are positioned in a way that is strictly dependent on the epigenome changes. Based on the presence of a specific epigenetic modification, the chromatin becomes less or more condensed. These changes in chromatin structure are inevitably related to gene activity. For example, DNA hypermethylation joined with histone hypoacetylation is frequently related to a condensed form of chromatin, marking the region of DNA that should not be active during a specific time window. This implies that genes in that specific region may become active once the aforementioned marks are removed. Indeed, epigenome represents a very powerful, extremely flexible "tool" for regulating gene activity and the major reason for the well-known phenomena of "time specific" and "tissue

In the field of cancer research, epigenome changes are considered to be among the first steps in carcinogenesis, preceding the structural changes in the DNA molecule, known generally as "gene mutations". Specifically, the most prominent change in the earliest phases of cancer is inactivation of tumor suppressor genes which are frequently silenced through DNA methyl‐ ation and histone deacetylation taking place in the regions corresponding to their promoters. It is known that enzymes regulating these processes, DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) are aberrantly up-regulated not only in a developed cancer, but also in the early phases of carcinogenesis (as recently shown for ductal *in situ* breast cancer [1]). Accordingly, significant effort has been given to the discovery and development of specific chemical compounds that may act as DNA demethylating agents and histone deacetylases

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Višnja Stepanić, Renata Novak Kujundžić and

Additional information is available at the end of the chapter

**Curcumin**

Koraljka Gall Trošelj

**1. Introduction**

specific" gene expression.

http://dx.doi.org/10.5772/58247
