Mechanisms controlling genomic imprinting and their dysregulation in human imprinting disorders.

Genomic imprinting is an epigenetic process that distinguishes parental alleles, resulting in parent-specific expression of a gene or cluster of genes. Imprints are acquired during gametogenesis when genome-wide epigenetic remodeling occurs. These imprints must then be maintained during preimplantation development, when another wave of genome-wide epigenetic remodeling takes place. Thus, for imprints to persist as parent-specific epigenetic marks, coordinated factors and processes must be involved to both recognize an imprint and protect it from genome-wide remodeling. Parent-specific DNA methylation has long been recognized as a primary epigenetic mark demarcating a genomic imprint. Recent work has advanced our understanding of how and when parent-specific DNA methylation is erased and acquired in the germ line as well as maintained during preimplantation development. Epigenetic factors have also been identified that are recruited to imprinted regions to protect them from genome-wide DNA demethylation during preimplantation development. Intriguingly, asynchrony in epigenetic reprogramming appears to be a recurrent theme with asynchronous acquisition between male and female germ lines, between different imprinted genes, and between the two parental alleles of a gene. Here, we review recent advancements and discuss how they impact our current understanding of the epigenetic regulation of genomic imprinting.

American College of Medical Genetics Statement on Diagnostic Testing for Uniparental Disomy

Is the risk of imprinting disorders increased in children conceived after ART? We found an adjusted odds ratio (AOR) of 2.84 [95% CI: 1.34-6.01] for Beckwith-Wiedemann syndrome in ART children, while the risk of Prader-Willi syndrome, Silver-Russell syndrome or Angelman syndrome was not increased in children conceived after ART. Earlier studies, most of them small, have suggested an association between ART and imprinting disorders. This was a binational register-based cohort study. All children conceived by ART in Denmark (n= 45 393, born between 1994 and 2014) and in Finland (n= 29 244, born between 1990 and 2014) were identified. The full background populations born during the same time periods in the two countries were included as controls. Odds ratios of imprinting disorders in ART children compared with naturally conceived (NC) children were calculated. The median follow-up time was 8years and 9months for ART children and 11years and 9months for NC children. From the national health registries in Denmark and Finland, we identified all children diagnosed with Prader-Willi syndrome (n= 143), Silver-Russell syndrome (n= 69), Beckwith-Wiedemann syndrome (n= 105) and Angelman syndrome (n= 72) born between 1994/1990 and 2014, respectively. We identified a total of 388 children diagnosed with imprinting disorders; 16 of these were conceived after ART. The overall AOR for the four imprinting disorders in ART children compared with NC children was 1.35 [95% CI: 0.80-2.29], but since eight ART children were diagnosed with Beckwith-Wiedemann syndrome, the AOR for this specific imprinting disorder was 2.84 [95% CI: 1.34-6.01]. The absolute risk of Beckwith-Wiedemann syndrome in children conceived after ART was still low: 10.7 out of 100 000 newborns. The risks of Prader-Willi syndrome, Silver-Russell syndrome and Angelman syndrome were not increased in children conceived after ART. Imprinting disorders are rare events and our results are based on few ART children with imprinting disorders. The aetiology is complex and only partly clarified, and the clinical diagnoses are challenged by a broad phenotypic spectrum. In the existing studies, results on the risk of imprinting disorders in children conceived after ART are ambiguous. This study adds that the risk of imprinting disorders in ART children is very small and perhaps restricted to Beckwith-Wiedemann syndrome. This work was supported by the Nordic Trial Alliance: a pilot project jointly funded by the Nordic Council of Ministers and NordForsk (grant number: 71450), the Nordic Federation of Obstetrics and Gynecology (grant numbers: NF13041, NF15058, NF16026 and NF17043) and the Interreg Öresund-Kattegat-Skagerak European Regional Development Fund (ReproUnion project). The authors have no conflicts of interest related to this work. N/A.

Imprinted genes play important roles in the regulation of growth and development, and several have been shown to influence behavior. Their allele-specific expression depends on inheritance from either the mother or the father, and is regulated by "imprinting control regions" (ICRs). ICRs are controlled by DNA methylation, which is present on one of the two parental alleles only. These allelic methylation marks are established in either the female or the male germline, following the erasure of preexisting DNA methylation in the primordial germ cells. After fertilization, the allelic DNA methylation at ICRs is maintained in all somatic cells of the developing embryo. This epigenetic "life cycle" of imprinting (germline erasure, germline establishment, and somatic maintenance) can be disrupted in several human diseases, including Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome (PWS), Angelman syndrome and Hydatidiform mole. In the neurodevelopmental Rett syndrome, the way the ICR mediates imprinted expression is perturbed. Recent studies indicate that assisted reproduction technologies (ART) can sometimes affect the epigenetic cycle of imprinting as well, and that this gives rise to imprinting disease syndromes. This finding warrants careful monitoring of the epigenetic effects, and absolute risks, of currently used and novel reproduction technologies.

BackgroundHuman-assisted reproductive technologies (ART) are a widely accepted treatment for infertile couples. At the same time, many studies have suggested the correlation between ART and increased incidences of normally rare imprinting disorders such as Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Silver-Russell syndrome (SRS). Major methylation dynamics take place during cell development and the preimplantation stages of embryonic development. ART may prevent the proper erasure, establishment, and maintenance of DNA methylation. However, the causes and ART risk factors for these disorders are not well understood.ResultsA nationwide epidemiological study in Japan in 2015 in which 2777 pediatrics departments were contacted and a total of 931 patients with imprinting disorders including 117 BWS, 227 AS, 520 PWS, and 67 SRS patients, were recruited. We found 4.46- and 8.91-fold increased frequencies of BWS and SRS associated with ART, respectively. Most of these patients were conceived via in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and showed aberrant imprinted DNA methylation. We also found that ART-conceived SRS (ART-SRS) patients had incomplete and more widespread DNA methylation variations than spontaneously conceived SRS patients, especially in sperm-specific methylated regions using reduced representation bisulfite sequencing to compare DNA methylomes. In addition, we found that the ART patients with one of three imprinting disorders, PWS, AS, and SRS, displayed additional minor phenotypes and lack of the phenotypes. The frequency of ART-conceived Prader-Willi syndrome (ART-PWS) was 3.44-fold higher than anticipated. When maternal age was 37 years or less, the rate of DNA methylation errors in ART-PWS patients was significantly increased compared with spontaneously conceived PWS patients.ConclusionsWe reconfirmed the association between ART and imprinting disorders. In addition, we found unique methylation patterns in ART-SRS patients, therefore, concluded that the imprinting disorders related to ART might tend to take place just after fertilization at a time when the epigenome is most vulnerable and might be affected by the techniques of manipulation used for IVF or ICSI and the culture medium of the fertilized egg.

Clinical Validation of a Genome-Wide DNA Methylation Assay for Molecular Diagnosis of Imprinting Disorders

Study question Is singleton birth from assisted reproductive technology (ART) associated with imprinting disorders (IDs) independent of parental infertility and other background factors? Summary answer Singletons conceived using intracytoplasmic sperm injection (ICSI) and frozen embryos have higher risks of Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome (PWS) and Silver-Russell syndrome (SRS). What is known already Previous studies have suggested elevated risk of IDs in children conceived with ART compared with children conceived with spontaneous conception (SC). It is not clear to what extent the observed associations could be explained by parental infertility and related risk factors. Knowledge on specific ART procedures and their association with IDs is also limited. Study design, size, duration A nationwide register-based cohort study was carried out in which all liveborn singletons in 1997-2017 in Sweden were included with follow-up to 2018. Participants/materials, setting, methods Among 1 998 825 singletons, 60 210 were conceived using ART. The International Classification of Diseases (ICD) version 10 was used to identify three distinct ID groups: PWS/SRS, BWS, and central precocious puberty (CPP). Cox regression, combined with inverse probability treatment weights to account for birth year, parity, pregnancy loss history, parental age and country of origin, was used to compare ART singletons with all SC singletons and those born to couples with known infertility. Main results and the role of chance A total of 1012 children were diagnosed with the IDs of interest (654 PWS/SRS, 255 BWS, and 109 CPP), and 49 of them were conceived through ART. Compared with all SC singletons, higher risk of PWS/SRS and BWS was observed in ART singletons and the weighted hazard ratios (wHRs) were 1.57 [95% CI, 1.09-2.26] and 2.57 [95% CI, 1.60-4.12], respectively. The elevated risks remained when comparison was restricted to SC singletons of couples with known infertility, though the wHR for BWS was somewhat attenuated (1.87, 95% CI: 1.04-3.36). No difference in risk of CPP was observed between singletons conceived with and without ART irrespective of parental infertility. Further subgroup analysis revealed that ICSI in combination with frozen embryo transfer was responsible for the higher risks of PWS/SRS (wHR 6.32, 95% CI: 3.34-11.95) and BWS (wHR 9.04, 95% CI: 4.34-18.82). Limitations, reasons for caution The use of the Swedish ICD-10 did not allow distinction of PWS and SRS. The number of CPP cases in ART singletons was too small (N = 3) to make inference. Wider implications of the findings This study found that ART-conceived singletons, particularly those conceived using ICSI and frozen embryos, had a higher risk of IDs independent of parental infertility. However, the underlying mechanism is not clear, and the role of type and severity of infertility warrants further investigation. Trial registration number not applicable

Epigenetics in Male Germ Cells

Genomic imprinting affects a subset of genes in mammals, such that they are expressed in a monoallelic, parent-of-origin-specific manner. These genes are regulated by imprinting control regions (ICRs), cis-regulatory elements that exhibit allele-specific differential DNA methylation. Although genomic imprinting is conserved in mammals, ICRs are genetically divergent across species. This raises the fundamental question of whether the ICR plays a species-specific role in regulating imprinting at a given locus. We addressed this question at the H19/insulin-like growth factor 2 (Igf2) imprinted locus, the misregulation of which is associated with the human imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). We generated a knock-in mouse in which the endogenous H19/Igf2 ICR (mIC1) is replaced by the orthologous human ICR (hIC1) sequence, designated H19(hIC1) We show that hIC1 can functionally replace mIC1 on the maternal allele. In contrast, paternally transmitted hIC1 leads to growth restriction, abnormal hIC1 methylation, and loss of H19 and Igf2 imprinted expression. Imprint establishment at hIC1 is impaired in the male germ line, which is associated with an abnormal composition of histone posttranslational modifications compared with mIC1. Overall, this study reveals evolutionarily divergent paternal imprinting at IC1 between mice and humans. The conserved maternal imprinting mechanism and function at IC1 demonstrates the possibility of modeling maternal transmission of hIC1 mutations associated with BWS in mice. In addition, we propose that further analyses in the paternal knock-in H19(+/hIC1) mice will elucidate the molecular mechanisms that may underlie SRS.

Genomic imprinting is an example of epigenetic inheritance in which differences in gene function depend on whether the allele was inherited from the mother or father. Many disorders appear to involve epigenetic alterations to imprinted genes. Genes involved in imprinting play important roles in both pre‐ and post‐natal growth and neurodevelopment. Imprinting disorders include several childhood genetic disorders such as Prader–Willi, Angelman and Beckwith–Wiedemann syndromes, as well as several types of cancer including Wilms tumour. Recent evidence suggests a correlation between assisted reproductive technologies (ART) and imprinting disorders, a phenomenon requiring more in‐depth studies as the prevalence of ART is increasing. The mechanism of normal imprinting as well as its perturbation in disease is becoming understood and amenable to experimentation. Key Concepts: Imprinted genes are expressed in a parent‐of‐origin‐dependent manner. Imprinting disorders result from genetic abnormalities in imprinted genes. Approximately 1% of the human genome is imprinted, a process which occurs during gamete development. Imprinted genes are generally associated with growth, neurodevelopment and epigenetics. The first imprinted genes discovered were IGF2 (paternally expressed) and H19 (maternally expressed), both located at 11p15. There is an imprinted gene cluster located at 15q11–q13, associated with Prader–Willi and Angelman syndromes. Imprinted genes/transcripts within this region include NDN , MAGEL2 , SNURF‐SNRPN , SNORD116 , SNORD115 and UBE3A . Well‐characterised imprinting disorders include Prader–Willi syndrome (PWS), Angelman syndrome (AS), Beckwith–Wiedemann syndrome (BWS), Albright hereditary osteodystrophy (AHO), pseudohypoparathyroidism (PHP), Silver–Russell syndrome (SRS) and transient neonatal diabetes mellitus (TNDM). Imprinted genes are associated with many types of cancers, primarily due to a process called loss of imprinting (LOI) which leads to aberrant gene expression. Cancers known to be associated with imprinted genes include Wilms tumour in children, and adult cancers of the ovary, lung, colon and liver. The relationship between artificial reproductive technology (ART) and imprinting disorders is currently being investigated. There is evidence to support a correlation between ART and imprinting disorders which result from loss of maternally imprinted genes such as AS and BWS.

Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS.

Assisted reproductive technologies (ARTs) have revolutionized infertility treatment, leading to the birth of over 10 million children worldwide. Despite their success, increasing concerns have been expressed regarding the potential long-term outcomes of ART-conceived individuals, particularly in relation to imprinting disorders (IDs). IDs result from the abnormal expression of imprinted genes, which are expressed in a parent-of-origin-specific manner and regulated by epigenetic mechanisms (e.g., DNA methylation). Disruption of these processes, through environmental, genetic, or procedural factors, can lead to disorders such as Beckwith–Wiedemann syndrome (BWS), Silver–Russell syndrome (SRS), Angelman syndrome (AS), and Prader–Willi syndrome (PWS). These syndromes are characterized by distinct clinical features, including growth abnormalities, neurodevelopmental delay, endocrine dysfunction, and cancer predisposition. ART procedures, especially ovarian hyperstimulation, in vitro fertilization (IVF), and embryo culture, coincide with critical periods of epigenetic reprogramming and may contribute to epimutations in imprinting control regions. In this review, we explored epidemiology, molecular mechanisms, and prenatal diagnostic strategies related to these four IDs in the context of ART. The findings suggest a higher prevalence of BWS and SRS in ART-conceived children. The data regarding AS and PWS are more controversial, with conflicting results across populations and methodologies. Although a causal link between ART and IDs remains debated, evidence suggests the potential contribution of ART procedures to epigenetic dysregulation in susceptible individuals. Further large-scale, methodologically rigorous studies will be essential to clarify this association and inform safer ART practices.

BackgroundImprinted genes, characterized by monoallelic expressions (either maternal or paternal), they are crucial for normal growth and development. Disruption of their monoallelic expressions leads to imprinting disorders (ImpDis). The aim of this study is to achieve proper diagnosis of ImpDis in Egyptian patients through clinical evaluation and genetic testing, emphasizing certain clinical manifestations that may indicate ImpDis to provide accurate diagnosis and genetic counseling.MethodsFifty-three patients, either clinically evaluated for Impaired Disposition (ImpDis) or suspected to have it, were referred from the outpatient genetic clinics at the National Research Center, Egypt. Nineteen patients displayed clinical manifestations of ImpDis syndromes, while 34 showed signs affecting growth, which suggested ImpDis. These growth-related symptoms included growth retardation, feeding problems, failure to thrive, hypoglycemia, obesity, hemihypertrophy, asymmetry, and overgrowth. Of the 19 patients with syndromic ImpDis, 8 were clinically diagnosed with Silver-Russell syndrome (SRS), 7 with Prader-Willi syndrome (PWS), and 4 with Beckwith-Wiedemann syndrome (BWS). We employed methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) for all patients, SNP-array testing for 12 patients, and whole exome sequencing (WES) for one patient.ResultsIn patients with Silver-Russell syndrome (SRS), one patient exhibited hypermethylation of the GRB10 and MEST genes, along with segmental uniparental disomy (UPD) on chromosome 7 (patient 1). Another patient had a variant in the HMGA2 gene (NM_001300918.1:c.310dup), which, according to the American College of Medical Genetics (ACMG) criteria, was classified as PM2 VUS (patient 2). In patients with Prader-Willi syndrome (PWS), one patient showed hypermethylation of the SNPRN gene (patient 3). In patients with Beckwith-Wiedemann syndrome (BWS), two displayed hypomethylation of the KCNQ-CR region (patients 4 and 5). Among the group of patients with symptoms suggestive of ImpDis, no methylation defects were detected through MS-MLPA.ConclusionIt is crucial to diagnose ImpDis accurately, as understanding the exact cause of ImpDis is important for genetic counseling and personalized medicine. Early diagnosis enables timely interventions, which can improve developmental outcomes. Precision in diagnosis helps differentiate between conditions with overlapping clinical features. HMGA2 mutation should be verified in SRs patients with negative 11p15 methylation defect and matUPD7.

In 2014, Facebook and Apple announced that they would pay for female employees to have their oocytes frozen to allow them to delay having children and instead focus on their careers. Whatever motivated the companies to make their offers, the fact that they did so highlights a prevalent problem faced by many young women: Their most fertile years are also a crucial period for building a career, when time off work may disadvantage them. > … cryopreservation is known to affect cell survival after thawing, which can have an impact on the subsequent clinical applications of frozen cells. To fulfill their offers, Facebook and Apple will need to offer their employees access to cryopreservation technologies that profoundly change the dynamics of family planning. Such technologies are not new, but work over the past decades has been aimed at increasing safety and efficacy and has reduced costs to the point that companies can now offer cryopreservation as a way to attract and retain female workers. Of course, the potential of cryopreservation goes far beyond freezing the eggs or sperm of ambitious young technology workers—it is a ubiquitous technology used in research and medicine for a wide variety of applications (Fig 1). For example, cryopreservation is used to store and transport biological material, including adult stem cells or stem cells from umbilical cord blood or bone marrow—both of which can later be used to treat disease or extend lifespan in the same patient—blood donations, especially of rare blood types, tissues, and organs. It is also offered as a crucial service for cancer patients to preserve their gametes before they undergo therapy that may render them infertile and, generally used in assisted reproduction to store oocytes, fertilized eggs, or embryos. Cryopreservation can contribute to environmental preservation efforts, where it is used to conserve the …

Fetal growth is a complex process depending on the genetics of the fetus, the availability of nutrients to the fetus, maternal nutrition and various growth factors and hormones of maternal, fetal and placental origin. The IGF system, and more particularly IGF2, is one of the most important endocrine and paracrine growth systems regulating fetal and placental growth (reviewed in [1]). The IGF2 gene is regulated by genomic imprinting and is expressed only from the paternally-inherited allele in most tissues during fetal development and after birth. Imprinted genes are tightly regulated and are therefore particularly susceptible to changes, including environmental and nutritional changes. Dysregulation of a cluster of imprinted genes, including the IGF2 gene within the 11p15 region, results in two fetal growth disorders (Silver-Russell and Beckwith-Wiedemann syndromes) with opposite growth phenotypes. Those two syndromes are model imprinting disorders to decipher the regulation of genomic imprinting.