Therapeutic Approaches to Imprinting Diseases

CHAPTER

THERAPEUTIC APPROACHES TO IMPRINTING DISEASES

28

Hiromitsu Hattori, Hitoshi Hiura, Norio Kobayashi, Souta Takahashi, Hiroaki Okae, Takahiro Arima Tohoku University Graduate School of Medicine, Sendai, Japan

CHAPTER OUTLINE 28.1 28.2 28.3 28.4

Introduction ...............................................................................................................................861 Assisted Reproductive Technologies and Imprinting Disorders.......................................................862 Clinical Management..................................................................................................................864 Future Perspective: Therapies .....................................................................................................867 28.4.1 Diagnostics ..........................................................................................................867 28.4.2 Interventions........................................................................................................867 28.4.2.1 Nutrition ....................................................................................................... 868 28.4.2.2 Stress Reduction........................................................................................... 868 28.4.2.3 Optimum Reproductive Age .......................................................................... 869 28.4.3 Therapies.............................................................................................................869 28.4.3.1 Drug Development ........................................................................................ 869 28.4.3.2 Induced Pluripotent Stem Cell Therapy.......................................................... 869 28.4.3.3 Gene Therapy ............................................................................................... 870 28.5 Conclusion.................................................................................................................................870 List of Abbreviations ............................................................................................................................871 Acknowledgments ................................................................................................................................872 References ..........................................................................................................................................872

28.1 INTRODUCTION Genomic imprinting is defined as parent-of-origin specific, monoallelic gene expression in mammals [1] and has essential roles in both early development and later life processes [2]. Differences in each parental genome are marked and established by DNA methylation during gametogenesis (DNA methylation is a covalent modification involving the transfer of a methyl group onto the cytosine to form 5-methylcytosine.). These DNA regions are named “germline differentially methylated regions (gDMRs)” and function as imprinting control regions (ICRs). The methylation marks are resisted and maintained despite the genome-wide demethylation in the blastocyst stage [3]. These gDMRs are characterized by differential epigenetic modifications that regulate chromatin at a cluster of imprinted genes, known as an imprinted domain (Epigenetics means that gene expression is controlled by Epigenetics in Human Disease. https://doi.org/10.1016/B978-0-12-812215-0.00028-5 Copyright © 2018 Elsevier Inc. All rights reserved.

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DNA methylation and histone modification without accompanying changes in DNA base sequence.). Furthermore, these gDMRs must be erased in primordial germ cells (PGCs) and reset for the next generation. Epigenetic errors in humans can cause congenital diseases for the next generation at any stage of the establishment, maintenance, or erasure of imprints. In mice, establishment of DNA methylation in the gDMR occurs in the growing oocyte in the diplotene stage of meiotic prophase I (female) and prospermatogonia gastrointestinal (GI) arrest (male). The key proteins in the establishment of the imprint machinery have been identified. Methylation imprints should be protected from genome-wide DNA demethylation (epigenetic reprogramming) and maintained by DNA methyltransferase (DNMT) and several proteins. Congenital imprinting disorders are normally rare childhood developmental disorders. Beckwithe Wiedemann syndrome (BWS: OMIM: 130650) and SilvereRussell syndrome (SRS: OMIM: 180860) are clinically distinct syndromes [4]. The former is a fetal overgrowth disorder in childhood. The etiology of this syndrome is associated mainly with epigenetic alterations on chromosome 11p15.5. The most frequent alteration observed in BWS is hypomethylation of the KCNQ1OT1 (LIT1) gDMR (>60% of BWS), which regulates the imprinted and tumor suppressor gene CDKNIC (P57Kip2). In other BWS, hypermethylation of H19 is observed and regulates the expression of the imprinted IGF2 gene. SRS is a clinically heterogeneous condition. The most frequent epigenetic alteration (epimutation) in SRS is hypomethylation of the H19 gDMR (40% of SRS) [5]. This gDMR regulates the expression of the imprinted IGF2 gene. Additional loci at chromosomes 7, 8, 15, 17, and 18 have also been implicated. Both these syndromes regulate the gene dosages at imprinted loci mediated mainly by epigenetic processes, which might consequently be subject to environmental influences, including assisted reproduction technology, on the epigenome [6] (see below). PradereWilli syndrome (PWS: OMIM: 176270) and Angelman syndrome (AS: OMIM: 105830) are also clinically distinct imprinting disorders linked to the same imprinted region on chromosome 15q11-q13. PWS is characterized by endocrine and neural abnormalities and malformation. This syndrome is mainly associated with maternal uniparental disomy (70%) and methylation error of paternal gDMR upstream of SNRPN gene (2%e5%). Recently, the snoRNAs (SNORD116) have been suggested to cause PWS via the modification of mRNA by alternative splicing and the snoRNA gene [7]. AS is characterized by global developmental delay, convulsions, scoliosis, excessive laughter, and movement, balance, and sleep disorders. This syndrome is associated with loss of function of the maternally expressed UBE3A gene through deletions (70%), paternal uniparental disomy (0%e20%), or aberrant methylation of gDMR upstream of SNRPN gene (2%e5%) of the maternal allele. UBE3A encodes ubiquitin-protein ligase E3A, which transfers ubiquitin from an E2 ubiquitin-conjugating enzyme. Several proteins (ECT2, P53, P27, HR23A, Arc, and ephexin-5) that are putatively targeted or modulated by ubiquitin-protein ligase E3A have been identified [8e11]. The deficits in mouse AS models include reduced spine density and lengths of certain neurons in the hippocampus, cortex, and cerebellum [12]. For both syndromes, epigenetic defects occurring less frequently have genetic alterations. The details of these and several other rare imprinting disorders are described elsewhere in this book.

28.2 ASSISTED REPRODUCTIVE TECHNOLOGIES AND IMPRINTING DISORDERS Epigenetic modifications in the germline provide a link between the environments, and alterations in gene expression lead to infertility and disease phenotypes. This suggests the strong possibility that the

28.2 ARTs AND IMPRINTING DISORDERS

863

use of assisted reproductive technologies (ARTs) to treat infertility may lead to disease. However, ARTs are a relatively recent technology and the longer-term consequences of ARTs treatments such as intracytoplasmic sperm injection (ICSI) and embryo freezing before transfer have not yet been manifested. It is still unknown when imprinting epigenetic errors related to human infertility arise and what factors may predispose to epigenetic changes. Hormonal stimulation of oocytes, in vitro culture, cryopreservation, and the timing of embryo transfer have all been shown to influence the proper establishment and maintenance of genomic imprints. Some infertile males, particularly those with oligozoospermia, carry preexisting imprinting errors in their sperm. Therefore, the process of ARTs and infertility itself might increase the risk of imprinting disorders [6]. A number of publications have suggested an association between ARTs and genomic imprinting disorders, especially BWS, SRS, and AS [13e18]. Many reports have suggested that imprint methylation errors occur at the time of DNA methylation reprogramming during the ARTs process both in in vitro fertilization (IVF) and ICSI [13,17e25] (Fig. 28.1). The exposure of gametes and early embryos to culture media and cryopreservation of embryos are also included [13,19,20,26]. Animal studies suggest that in vitro culture of embryos to the blastocyst stage may be associated with epimutations. Large offspring syndrome in

ICSI Cryopreservation

ROSI PGC

Dnmt3a Dnmt3L Dnmt3b

Sperm

Methylation

High

Dnmt1o Dnmt1s Dppa3 (Pgc7/Stella) Zfp57

Embryonic tissue

Blastocyst

Extraembryonic tissue

Low

Dnmt3a Dnmt3L Kdm1b Zfp57 Transcription

Oocyte

Fertilization

Gonadotropins Ooplasmic transter IVM

Implantation IVF Extended culture Cryopreservation

FIGURE 28.1 Imprints in gametogenesis and embryogenesis. During the transition from primordial to antral follicles in the postnatal growth phase (postpachytene), methylation is acquired asynchronously in a gene-specific manner in mouse oogenesis. In sperm, imprint methylation is initiated prenatally before meiosis and is completed by the pachytene phase of postnatal spermatogenesis. The imprints of gametes are maintained stably in the early embryo despite overall epigenetic reprogramming. ARTs result from the use of sperm with incomplete reprogramming and from in vitro embryo procedures performed at the time of epigenetic reprogramming. ART, assisted reproductive technology; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; IVM, in vitro oocyte maturation; PGC, primordial germ cell; ROSI, round spermatid injection.

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CHAPTER 28 THERAPEUTIC APPROACHES TO IMPRINTING DISEASES

male cattle undergoing ARTs is associated with loss of maternal allelic methylation in the Igf2R gDMR [27,28] and has phenotypic similarity to BWS. Blastocyst transfer and embryonic cryopreservation have also been found to have deleterious effects on DNA methylation [29,30]. Case reports of monochorionic dizygotic twins and conjoined twins with BWS resulting from transfer at the longcultured blastocyst stage [31,32] showed demethylation of KCNQ1OT1 (LIT1). In addition to epimutations after ARTs, there is evidence that sperm and oocytes from patients with fertility issues carry preexisting epigenetic errors. At spermatogenesis, the genome undergoes major changes that influence epigenetic information. Azoospermia and oligozoospermia caused by secondary inflammatory obstruction are related to a gain in DNA methylation at maternal gDMRs, which are unmethylated in sperm. Altered methylation in sperm from infertile male may related to the improper erasure of DNA methylation in PGCs of many nonimprinted genes, in addition to abnormal methylation levels in gDMRs [33]. Detailed analysis of abnormal methylation patterns in imprinting disorders may provide clues as to the causes of these diseases. In some patients with BWS and SRS, multilocus methylation defects (MLMDs) are more likely to be affected in babies conceived after ARTs than in those conceived spontaneously [34]. However, no correlation has been observed between clinical severity and either the degree of methylation loss or the MLMDs.

28.3 CLINICAL MANAGEMENT The treatment of the imprinting disorders is mainly maintenance therapy. Details of the diagnosis and therapy are described elsewhere in detail (Nature Reviews 2017, GeneReviews 2016). Here, we will introduce the main clinical features and the management of four representative imprinting disorders (Table 28.1). BWS is an overgrowth disorder characterized by neonatal hypoglycemia, macrosomia, macroglossia, hemihyperplasia, omphalocele, and embryonal tumors. Early death may occur from complications of hypoglycemia, cardiomyopathy, macroglossia, or malignant tumors. Macroglossia and macrosomia are generally present at birth. The growth rate slows down in childhood. For the neonate, treatment of hypoglycemia is important to reduce the risk of central nervous system complications. Omphalocele of the abdominal wall should also be repaired, and endotracheal intubation for a compromised airway and the use of specialized nipples or nasogastric tube feeding are needed to manage feeding difficulties resulting from macroglossia. In infancy or early childhood, children with macroglossia are treated via tongue reduction surgery and speech therapy. During early puberty, surgery may be performed to equalize significant differences in leg length secondary to hemihyperplasia, and craniofacial surgery may be of benefit to those with facial hemihyperplasia. Embryonic tumors are treated according to the standard protocols. Nephrocalcinosis and other renal findings should be assessed and treated by a pediatric nephrologist (www.ncbi.nlm.nih.gov). SRS is characterized clinically by growth failure, severe feeding difficulties, GI problems, hypoglycemia, body asymmetry, scoliosis, motor and speech delays, and psychosocial challenges. For the neonate, adequate nutritional status is important with the awareness that rapid postnatal weight gain might lead to a subsequent increased risk of metabolic disorders. Patients with SRS are treated with growth hormone to improve body composition, motor development, and appetite, as well as to reduce the risk of hypoglycemia and to increase height. Clinicians should be aware of possible premature adrenarche, fairly early and rapid puberty, and insulin resistance. Treatment with

28.3 CLINICAL MANAGEMENT

865

Table 28.1 Clinical Management of Imprinting Disorders

BWS

Clinical Symptom

Management

· Hypoglycemia · Omphalocele · Macrosomia

· To reduce the risk of central nervous system complications · Abdominal wall repair for omphalocele soon after birth of difficulties with endotracheal intubation. Assessment of · Anticipation respiratory function, possibly including sleep study to address concern regarding potential sleep apnea.

· Management of feeding difficulties using specialized nipples such as · · Cleft palate · Craniofacial surgeon · Orthopedic surgeon · Neoplasias of the · Anomalies renal tract · GI tract abnormalities · Cardiac problems · Others

· · · · · · · · ·

SRS

· Postnatal growth failure

· ·

age advancement · Bone and puberty metabolic · Long-term complications

· · ·

the longer nipple recommended for babies with cleft palate or, rarely, short-term use of nasogastric tube feedings Tongue growth does slow over time and jaw growth can accelerate to accommodate the enlarged tongue. Some children benefit from tongue reduction surgery Management of cleft palate following standard protocols Referral to a craniofacial surgeon if facial hemihyperplasia is significant Consultation with an orthopedic surgeon if hemihyperplasia results in a significant difference in leg length Treatment of neoplasias following standard pediatric oncology protocols Developmental anomalies of the renal tract are associated with increased calcium excretion and deposition Referral of children with structural GI tract abnormalities to the relevant specialist Management of cardiac problems following standard protocols Screening for embryonal tumors, which has traditionally involved the following. Abdominal ultrasound examination every 3 months until age 8 years Measurement of serum alpha-fetoprotein concentration every 2e3 months in the first 4 years of life for early detection of hepatoblastoma The first 2 years of life are nutritional support, prevention of hypoglycaemia, and recovery of any calorie-related length or height deficit, which should be addressed before initiation of GH therapy Careful monitoring is needed, especially during nonvolitional feeding, because rapid catch-up weight gain in children born SGA has been associated with an increased risk of metabolic and cardiovascular disease in later life Early bone age delay is followed by rapid advancement typically at around 8e9 years of age Onset of puberty is usually within the normal range (8e13 years in girls and 9e14 years in boys) at the younger end of the spectrum A low birth weight infant is at increased risk of adult health problems, including coronary heart disease hypertension, dyslipidemia, insulin resistance, and obesity (the metabolic syndrome). Infant born SGA indicate that those who have rapid or disproportionate catch-up in weight are at particularly high risk. Continued

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Table 28.1 Clinical Management of Imprinting Disordersdcont’d Clinical Symptom

· Neurocognitive problems

· Orthopedic problems ·

Maxillofacial abnormalities

congenital · Other anomalies

PWS

· Feeding · Hyperphagia · Obesity

Management

delay might be related to reduced muscle bulk and fairly large · Motor head size · Verbal dyspraxia and more global developmental delay or learning difficulties, usually mild. Autistic spectrum disorder is more frequent problems seen include limb or body asymmetry, scoliosis, · Orthopedic hip dysplasia, and hand and/or foot anomalies. Limb asymmetry can

· · · · · · · ·

AS

· Education · Behavior management · Hypogonadism · Sleep issues · Skin picking problems · Feeding in newborns · Seizure · Hypermotoric behaviors

· · · · · · · ·

affect the arms, legs, or both Craniofacial disproportion, which results in a triangular-shaped face Delayed dental eruption, microdontia, absence of secondary teeth, and blunted condyles Genital abnormalities, including cryptorchidism and hypospadias, occur frequently in boys MayereRokitanskyeKusterfemale patients is characterized by congenital hypoplasia or aplasia of the uterus and upper part of the vagina Structural renal anomalies and congenital heart defects Special feeding techniques, including special nipples or gavage feeding, may be necessary for the first weeks to months of life A program of a well-balanced, low-calorie diet, regular exercise, and close supervision to minimize food stealing should be instituted to prevent obesity Assessment of adequacy of vitamin and mineral intake by a dietician and prescription of appropriate supplementation are indicated, especially for calcium and vitamin D Growth hormone treatment normalizes height, increases lean body mass, decreases fat mass, and increases mobility Begin speech therapy for language delay and articulation abnormalities in infancy and childhood Cryptorchidism may resolve spontaneously, even up to adolescence, but usually requires hormonal and surgical approaches Disturbed sleep in children and adults should prompt a sleep study Decreased skin picking with topiramate treatment in some individuals Require special nipples and other strategies to manage weak or uncoordinated sucking Many antiepileptic drugs have been used to treat seizures Typically resistant to behavioral therapies

AS, Angelman syndrome; BWS, BeckwitheWiedemann syndrome; GH, growth hormone; GI, gastrointestinal; PWS, PradereWilli syndrome; SGA, small for gestational age; SRS, SilvereRussell syndrome.

28.4 FUTURE PERSPECTIVE: THERAPIES

867

gonadotropin-releasing hormone analogs can delay the progression of central precocious puberty and preserve adult height potential (www.ncbi.nlm.nih.gov). PWS is characterized by severe hypotonia and feeding difficulties in early infancy, followed in later infancy or early childhood by excessive eating and gradual obesity. Motor and language development are delayed. All individuals have some degree of cognitive impairment. Hypogonadism is present as genital hypoplasia, incomplete pubertal development, and, in most, infertility. Short stature is common, and scoliosis is often present. In infancy, a special nipple or enteral tube feeding is needed to assure adequate nutrition. Physical therapy may improve muscle strength, and hormonal and surgical treatments can be considered for cryptorchidism. In childhood, strict supervision of daily food intake based on height, weight, and the body mass index (BMI) is necessary to provide energy requirements while limiting excessive weight gain and encouraging physical activity. Growth hormone replacement therapy is implemented to normalize height, increase the lean body mass and mobility, and decrease the fat mass. Replacement of sex hormones at puberty produces adequate secondary sexual characteristics (www.ncbi.nlm.nih.gov). AS is characterized by severe developmental delay or intellectual disability, severe speech impairment, gait ataxia and/or tremulousness of the limbs, and a unique behavior with an inappropriate happy demeanor that includes frequent laughing, smiling, and excitability. Microcephaly and seizures are also common. Developmental delays are first noted at around the age of 6 months; however, the unique clinical features of AS do not become manifest until after the age of 1 year, and it can take several years before the correct clinical diagnosis is obvious. The disease is dealt with routine management of feeding difficulties, constipation, gastroesophageal reflux, and strabismus. Antiepileptic drugs are used for seizures. In addition, patients need physical therapy, occupational therapy, and speech therapy with an emphasis on nonverbal methods of communication, including augmentative communication aids and gesturing (www.ncbi.nlm.nih.gov).

28.4 FUTURE PERSPECTIVE: THERAPIES 28.4.1 DIAGNOSTICS ARTs can be used for preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). This diagnosis can provide key information on imprinting disorders. PGD is usually used to test a single embryo gene for a distinct pathologic condition, whereas PGS is a screening test offered to couples to detect chromosomal translocations, mitochondrial diseases, late-onset autosomal dominant diseases, and aneuploidy [35]. Currently, PGD or PGS is indicated in couples who have a history of multiple spontaneous abortions, a family history of X-linked disease or certain single-gene diseases, and advanced maternal age. PGD and PGS use cells from embryos or oocytes before embryo implantation. Frozen embryo transfer is a well-established method employed throughout the world. A frozen embryo after IVF and ICSI developed to a blastocyst could be of benefit for preimplantation methylation screening to reduce the risk of imprinting disorders. However, there have as yet been no reports on this technique.

28.4.2 INTERVENTIONS While the plasticity of epigenetic marks such as DNA methylation and histone modification may make these more susceptible to modification by ARTs (Fig. 28.1) environmental factors and diet (Fig. 28.2),

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CHAPTER 28 THERAPEUTIC APPROACHES TO IMPRINTING DISEASES

Folic acid Methionine

THF Vitamin B6

Betaine-homocysteine methyltrasferase

Unmethylated compounds

choline

Methionine synthase

5, 10Vitamin B12 CH3-THF

SAM

Betaine SAH

5-CH3-THF

Methylated compounds (e.g., creatine)

SAH hydrolase

Homocysteine

FIGURE 28.2 The methionineehomocysteine cycle. The enzymes are in italics and the cofactor is in black box. The compounds that were administered in the two clinical trials involving “promethylation” compounds, as described in the text, are shown in the boxes. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; THF, tetrahydrofolate.

this also suggests that aberrant epigenetic marks may be reversible. A greater understanding of this process and the function of methylation-modifying drugs may lead to the development of new treatment methods. Clinical trials involving oral administration of folate, vitamin B12, creatine, and betaine have been undertaken in an attempt to augment DNA methylation pathways. The availability of specific nutrients, alterations in the expression, localization and activity of epigenetic modifiers such as the DNMTs, the histone-modifying enzymes, and their associated proteins may play a role in the aberrant epigenome in germ cells. Some modifiers are specifically expressed in germ cells and play crucial roles in germ cellespecific genes such as Dnmt3L and Prdm9. Consequently dietary-driven alterations in epigenetic regulators can lead to the development of poorquality germ cells and may cause imprinting disorders.

28.4.2.1 Nutrition There are a number of causal environmental factors for epimutations in germ cells and early embryos. Nutrition may alter DNA methylation because one-carbon metabolism is dependent on dietary methyl donors and on the cofactors methionine, choline, folic acid, and vitamin B12 [36]. The limited availability of acetyl-CoA for histone acetyltransferase activity and methyl donors of S-adenosylmethionine provided via the folate-methionine pathway may therefore play an important role in the establishment and maintenance of inappropriate epigenetic patterns (Fig. 28.2). Conversely, dietary supplementation may also provide a route to attenuating inappropriate DNA methylation patterns resulting from a decrease in DNMT1 activity, which can be changed in the germline and early embryo [6].

28.4.2.2 Stress Reduction Social stress acting through hormone signaling pathways is known to influence epigenetic modifications. The extent and type of maternal care very early in life have been shown to affect epigenetic marks at the glucocorticoid receptor in the neonatal hippocampus and influence later life stress

28.4 FUTURE PERSPECTIVE: THERAPIES

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responses in the offspring [37,38]. Furthermore, psychosocial stress, comparable with human posttraumatic stress disorder (PTSD), leads to an increase in brain-derived neurotrophic factor (BDNF) methylation in the dorsal hippocampus and downregulation of BDNF expression in the dorsal and ventral hippocampus. The induction of region-specific epigenetic changes in response to traumatic stress demonstrates that DNA methylation remains an active process that can be shaped by environmental factors even in the nervous system. The effect of stress on the imprint methylation on germ cells and early embryos has not been investigated. However, such stress is also a cause of male or female infertility [39,40], which may occur through epigenetic alterations in the germline and embryo. Endocrine disruptors are also potential environmental factors driving abnormal germ cell development. Seminal tract infection, one of the most common causes of infertility in men, may also contribute to abnormal spermatogenesis and epigenetic errors.

28.4.2.3 Optimum Reproductive Age Aging is also associated with germline epigenetic changes. Recent reports have identified ageassociated sperm DNA methylation alterations [41]. Interestingly, some of the age-related changes in sperm DNA methylation are located in genes previously associated with schizophrenia and bipolar disorder. In general, the sperm of aged men are affected by the men’s accumulated exposures to environmental and nutritional factors such as reproductive toxicants, certain foods, and drug exposures during gametogenesis.

28.4.3 THERAPIES 28.4.3.1 Drug Development Patients with PWS and AS are mainly treated via gene target therapy, whereas patients with BWS and SRS undergo epigenetic therapy. For PWS and AS, gene target therapy may be effective. Recent therapeutic efforts have focused on activating the silenced paternal UBE3A allele via use of topoisomerase inhibitors [42] and antisense oligonucleotides (ASOs) [43]. In mice, a successful attempt was made to activate the paternal Ube3a via use of the topoisomerase inhibitor topotecan [42]. However, this is an anticancer drug and is very toxic to normal tissues. It has been shown to interfere not only with the target gene, small nucleolar RNA host gene 14 (SNHG14, which functions as an antisense UBE3A transcript), but also with long transcripts in general [44]. Another approach is based on RNA interference by ASOs against SNHG14, which leads to degradation of this transcript. ASOs were found to have long-term effects in an AS mouse model [43]. This approach is now being further developed by a pharmaceutical company, which is planning to start a phase I clinical trial [45]. However, all clinical trials in humans have been unsuccessful and have not improved neurodevelopment in AS. Topoisomerase inhibitors and ASOs are being developed to directly inhibit UBE3A-AS using cultured human neurons [44,46,47].

28.4.3.2 Induced Pluripotent Stem Cell Therapy Induced pluripotent stem (iPS) cells may also be available for treatment. Recently, it was reported that the expression state of the imprinted Dlk1-Dio3 cluster on mouse chromosome 12 is often altered in iPS cells [48]. In the affected mouse iPS cell clones, the ICR within the cluster is abnormally methylated and associated imprinted genes such as Meg3 are abnormally silenced. Furthermore, these iPS cell clones contribute poorly to chimeras and fail to support the development of entirely iPS

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CHAPTER 28 THERAPEUTIC APPROACHES TO IMPRINTING DISEASES

cellederived mice, whereas embryos derived from iPS cell clones with normal Meg3 expression develop well [48]. Clone- and gene-specific aberrations of imprinting were also reported in human iPS cells [49]. These studies underscore the importance of the imprint maintenance mechanism in cell reprogramming. The iPS cells of patients are useful for biological analyses of imprinting diseases. Normal iPS cells may be replaced because of lack of function to maintain the normal function of nerve cells.

28.4.3.3 Gene Therapy Recently, artificial transcription factors (ATFs) were developed to activate UBE3A and inhibit UBE3A-AS. ATFs are synthetics that regulate transcription of genes by binding to specific target DNA sequences, after which ATF-binding proteins are guided into the nucleus. The DNA-binding protein may be a zinc finger protein (ZFP), transcription activator-like effector [50], or a clustered regularly interspaced short palindromic repeat (CRISPR)ederived protein. ZFP-ATFs are being developed for the treatment of Huntington disease, retinitis pigmentosa [51], amyotrophic lateral sclerosis, and diabetic neuropathy. ZFP-ATFs targeting UBE3A and its regulatory regions are being developed with a mixture of activation and repressor domains [52]. The ATF with the strongest activity is anticipated to be used for the treatment of PWS or AS [53]. Several other radical therapies are still under investigation and no conclusions regarding their long-term safety and efficacy can be drawn as yet [54].

28.5 CONCLUSION Finally, recent whole genome methylation analyses of human germ cells and early embryos have revealed similarities and differences in the DNA methylation dynamics of human and mouse embryos [55e57]. The human sperm genome is predominantly hypermethylated, except for CpG islands (CGIs), as is the mouse sperm genome. Similar to mouse oocytes, human oocytes have large hyperand hypomethylated domains associated with transcription. However, the methylated regions are substantially different in human and mouse oocytes, which may reflect their divergent transcriptome profiles [58]. Furthermore, we found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts (Fig. 28.3). Remarkably, oocyte-specific methylated CGIs show methylation levels very similar to known gDMRs in human blastocysts. ICRs are resistant to demethylation after fertilization, and many oocyte-specific methylated CGIs may remain maternally methylated in human blastocysts. Consistently, recent studies have revealed that many of the oocyte-specific methylated CGIs actually serve as ICRs in the human placenta [59e61]. Most of these novel ICRs do not maintain maternal methylation in somatic cells. However, the associations of these ICRs and human diseases are still unknown. Here, we have summarized the current knowledge on the therapeutic approaches to four imprinting disorders. However, there have not yet been any successful clinical trials for humans, though there have been various attempts to develop drugs using mouse models. Several environmental factors are thought to be the causal factors for epigenetic alterations. The use of ARTs may be one of the contributory factors, but there is as yet no direct evidence. A greater understanding of the plasticity of epigenetic marks in germ cells and embryos will lead to the development of epidrugs and new treatment methods in the future.

LIST OF ABBREVIATIONS

(B)

Methylation

Methylation

(A)

871

Fertilization

Implantation

Fertilization

Implantation

FIGURE 28.3 DNA methylation dynamics in early human and mouse embryos. (A) Schematic illustration of DNA methylation dynamics during early mouse development. After fertilization, both maternal and paternal genomes are demethylated followed by de novo methylation after implantation. Ten-eleven translocationeindependent active demethylation and de novo methylation of the paternal genome in zygotes are omitted. (B) Schematic illustration of DNA methylation dynamics during early human development. The maternal genome is demethylated to a much lesser extent than the paternal genome in human preimplantation embryos.

LIST OF ABBREVIATIONS ART AS ASOs ATFs BDNF BMI BWS CGIs CRISPR DNMT gDMRs GI ICRs ICSI iPS IVF MLMDs OMIM PGCs PGD PGS PTSD PWS SNHG14 SRS ZFP

Assisted reproductive technology Angelman syndrome Antisense oligonucleotides Artificial transcription factors Brain-derived neurotrophic factor Body mass index BeckwitheWiedemann syndrome CpG islands Clustered regularly interspaced short palindromic repeat DNA methyltransferase Germline differentially methylated regions Gastrointestinal Imprinting control regions Intracytoplasmic sperm injection Induced pluripotent stem In vitro fertilization Multilocus methylation defects Online mendelian inheritance in man Primordial germ cells Preimplantation genetic diagnosis Preimplantation genetic screening Posttraumatic stress disorder PradereWilli syndrome Small nucleolar RNA host gene 14 SilvereRussell syndrome Zinc finger protein

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CHAPTER 28 THERAPEUTIC APPROACHES TO IMPRINTING DISEASES

ACKNOWLEDGMENTS We thank all the members of our laboratory for their valuable suggestions. This work was partially supported by a Grant-in-Aid for project from the Japan Agency for Medical Research and Development (AMED), 17ek0109101h0003, 17ek0109132h0003.

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