Yuk Jing Loke

The role of epigenetic change in autism spectrum disorders

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders characterized by problems with social communication, social interaction, and repetitive or restricted behaviors. ASD are comorbid with other disorders including attention deficit hyperactivity disorder, epilepsy, Rett syndrome, and Fragile X syndrome. Neither the genetic nor the environmental components have been characterized well enough to aid diagnosis or treatment of non-syndromic ASD. However, genome-wide association studies have amassed evidence suggesting involvement of hundreds of genes and a variety of associated genetic pathways. Recently, investigators have turned to epigenetics, a prime mediator of environmental effects on genomes and phenotype, to characterize changes in ASD that constitute a molecular level on top of DNA sequence. Though in their infancy, such studies have the potential to increase our understanding of the etiology of ASD and may assist in the development of biomarkers for its prediction, diagnosis, prognosis, and eventually in its prevention and intervention. This review focuses on the first few epigenome-wide association studies of ASD and discusses future directions.

Cohort Profile: The Peri/post-natal Epigenetic Twins Study

Cancer and Disease Epigenetics Group, Murdoch Childrens Research Institute (MCRI), Royal Children’s Hospital, Parkville, Victoria, Australia, Early Life Epigenetics Group, MCRI, Royal Children’s Hospital, Parkville, Victoria, Australia, Clinical Epidemiology and Biostatistics Unit, MCRI, Royal Children’s Hospital, Parkville, Victoria, Australia, Department of Paediatrics, University of Melbourne, Victoria, Australia, The Ritchie Centre, Monash Institute of Medical Research, Victoria, Australia, Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women’s Hospital, Melbourne, Victoria, Australia, Department of Obstetrics and Gynaecology,University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia Present address: Department of Public Health, Twin Study, Hjelt Institute, University of Helsinki, Finland

An epigenetic clock for gestational age at birth based on blood methylation data

BackgroundGestational age is often used as a proxy for developmental maturity by clinicians and researchers alike. DNA methylation has previously been shown to be associated with age and has been used to accurately estimate chronological age in children and adults. In the current study, we examine whether DNA methylation in cord blood can be used to estimate gestational age at birth.ResultsWe find that gestational age can be accurately estimated from DNA methylation of neonatal cord blood and blood spot samples. We calculate a DNA methylation gestational age using 148 CpG sites selected through elastic net regression in six training datasets. We evaluate predictive accuracy in nine testing datasets and find that the accuracy of the DNA methylation gestational age is consistent with that of gestational age estimates based on established methods, such as ultrasound. We also find that an increased DNA methylation gestational age relative to clinical gestational age is associated with birthweight independent of gestational age, sex, and ancestry.ConclusionsDNA methylation can be used to accurately estimate gestational age at or near birth and may provide additional information relevant to developmental stage. Further studies of this predictor are warranted to determine its utility in clinical settings and for research purposes. When clinical estimates are available this measure may increase accuracy in the testing of hypotheses related to developmental age and other early life circumstances.

Association of in vitro fertilization with global and IGF2 / H19 methylation variation in newborn twins

In vitro fertilization (IVF) and its subset intracytoplasmic sperm injection (ICSI), are widely used medical treatments for conception. There has been controversy over whether IVF is associated with adverse short- and long-term health outcomes of offspring. As with other prenatal factors, epigenetic change is thought to be a molecular mediator of any in utero programming effects. Most studies focused on DNA methylation at gene-specific and genomic level, with only a few on associations between DNA methylation and IVF. Using buccal epithelium from 208 twin pairs from the Peri/Postnatal Epigenetic Twin Study (PETS), we investigated associations between IVF and DNA methylation on a global level, using the proxies of Alu and LINE-1 interspersed repeats in addition to two locus-specific regulatory regions within IGF2/H19, controlling for 13 potentially confounding factors. Using multiple correction testing, we found strong evidence that IVF-conceived twins have lower DNA methylation in Alu, and weak evidence of lower methylation in one of the two IGF2/H19 regulatory regions and LINE-1, compared with naturally conceived twins. Weak evidence of a relationship between ICSI and DNA methylation within IGF2/H19 regulatory region was found, suggesting that one or more of the processes associated with IVF/ICSI may contribute to these methylation differences. Lower within- and between-pair DNA methylation variation was also found in IVF-conceived twins for LINE-1, Alu and one IGF2/H19 regulatory region. Although larger sample sizes are needed, our results provide additional insight to the possible influence of IVF and ICSI on DNA methylation. To our knowledge, this is the largest study to date investigating the association of IVF and DNA methylation.

Quantitation of the cellular content of saliva and buccal swab samples

Buccal swabs and saliva are the two most common oral sampling methods used for medical research. Often, these samples are used interchangeably, despite previous evidence that both contain buccal cells and blood leukocytes in different proportions. For some research, such as epigenetic studies, the cell types contributing to the analysis are highly relevant. We collected such samples from twelve children and twenty adults and, using Papanicolaou staining, measured the proportions of epithelial cells and leukocytes through microscopy. To our knowledge, no studies have compared cellular heterogeneity in buccal swab and saliva samples from adults and children. We confirmed that buccal swabs contained a higher proportion of epithelial cells than saliva and that children have a greater proportion of such cells in saliva compared to adults. At this level of resolution, buccal swabs and saliva contained similar epithelial cell subtypes. Gingivitis in children was associated with a higher proportion of leukocytes in saliva samples but not in buccal swabs. Compared to more detailed and costly methods such as flow cytometry or deconvolution methods used in epigenomic analysis, the procedure described here can serve as a simple and low-cost method to characterize buccal and saliva samples. Microscopy provides a low-cost tool to alert researchers to the presence of oral inflammation which may affect a subset of their samples. This knowledge might be highly relevant to their specific research questions, may assist with sample selection and thus might be crucial information despite the ability of data deconvolution methods to correct for cellular heterogeneity.

Identical twins doubly exchanged at birth: a case report of genetic and environmental influences on the adult epigenome

AIM Epigenetic comparisons within monozygotic twin pairs have enhanced our understanding of nongenetic mechanisms underlying disease etiology. We present epigenetic findings for a unique case of doubly exchanged Colombian male monozygotic twins raised in extremely different environments. RESULTS Using genome-wide DNA methylation data from cheek swabs from which blood-specific differentially methylated probes had been removed, the individuals grouped by shared genetics rather than shared environment, except for one twin who presented as an outlier. Closer inspection of DNA methylation differences within both reared-apart twin pairs revealed several genes and genetic pathways likely to be influenced by the rearing environment. CONCLUSION Together with our previous findings, we suggest that genetics, pre- and postnatal environments contribute to the epigenetic profile, although additional studies are needed to quantify these effects.

Are the effects of IVF on DNA methylation driven by intracytoplasmic sperm injection and male infertility

The term ‘in vitro fertilization’ (IVF) covers a number of related techniques in which an oocyte is joined with a sperm in vitro to fuse and form a zygote, which is later transferred to the woman’s uterus, where it develops as an embryo. Here we briefly review what is known about the developmental and epigenetic consequences of IVF, then we focus on the specific question of whether the effects of IVF on DNA methylation could be driven by procedure of intracytoplasmic sperm injection (ICSI), used in a subset of IVFs. IVF procedures have been around for almost 40 years and are widespread throughout the developed world, contributing to between 0.5 and 5% of live births [1]. Most IVF protocols involve ovarian stimulation, sperm and oocyte retrieval and preparation (including sperm washing), fertilization via coincubation, embryo culture and embryo selection and transfer [2]. There are many variations in IVF methods including cryopreservation of the embryo prior to transfer, assisted hatching and extended culture prior to transfer. In ICSI, sperm injection replaces coincubation. In general, IVF has been linked with a slightly higher risk of birth defects such as spina bifida, cerebral palsy and abnormalities of the cardiovascular and musculoskeletal systems [3]. Some have found that such effects disappeared after controlling for other IVFassociated factors [4]. Others have linked IVF with a larger placental/birth weight ratio and higher levels of childhood cardiometabolic risk factors such as blood pressure and fasting glucose [5]. A recent meta-analysis of four studies found an almost fourfold incidence in imprinting disorders such as Angelman syndrome, Beckwith–Wiedemann syndrome and Prader–Willi syndrome, in IVF-conceived versus naturally conceived children [6]. Many animal and human studies have attempted to separate the effects of specific stages of IVF [7]. Culture media components such as oxygen concentration and amino acid composition affect embryo viability and although many studies have linked some culture media to low birth weight and adverse neurodevelopmental outcomes, others have not. One recent study of 73 IVFs randomized to different culture media showed that the choice of media can have an effect on neurodevelopmental outcomes in childhood [8]. In addition, extended culture prior to embryo transfer has been associated with an increased incidence of preterm birth but cryopreservation of embryos prior to transfer seems to avoid such risks. We next consider whether IVF procedures interfere with epigenetic remodeling in early development. Between fertilization and implantation, most gamete-specific epigenetic marks, including DNA methylation, are removed [9]. Following implantation, levels of DNA methylation rise as tissue-specific epigenetic marks are established. In imprinted genes, expressed in somatic cells from a single parental allele, things are a little different. Epigenetic state at imprinting control “...evidence tells us that both genetic and epigenetic defects could be inherited from infertile men via intracytoplasmic sperm injection.”

Relationship between Epigenetic Maturity and Respiratory Morbidity in Preterm Infants

Objective To assess associations between epigenetic maturity of extremely preterm babies (born at less than 28 weeks of gestation), neonatal interventions, and respiratory outcomes, including the administration of surfactant and postnatal corticosteroids, duration of assisted ventilation, and development of bronchopulmonary dysplasia (BPD). Study design DNA was extracted from neonatal blood spots collected after birth from 143 extremely preterm infants born 1991‐1992 in Victoria, Australia and used to determined DNA methylation (DNAm). A DNAm based gestational age was determined using our previously published method. The residual of DNAm gestational age and clinically estimated gestational age (referred to as “gestational age acceleration”) was used as a measure to assess developmental maturity. Associations between gestational age acceleration and respiratory interventions and morbidities were determined. Results Infants with higher gestational age acceleration were less likely to receive surfactant (P = .009) or postnatal corticosteroids (P = .008), had fewer days of assisted ventilation (P = .01), and had less BPD (P = .02). Respiratory measures are known to correlate with gestational age; however, models comparing each with clinically estimated gestational age were improved by the addition of the gestational age acceleration measure in the model. Conclusions Gestational age acceleration correlates with respiratory interventions and outcomes of extremely preterm babies. Surfactant and postnatal corticosteroid use, assisted ventilation days, and BPD rates were all lower in babies who were epigenetically more mature than their obstetrically estimated gestational age. This suggests that gestational age acceleration is a clinically relevant metric of developmental maturity.