Epigenomics: Beyond Genome Sequencing

Epigenomics: Beyond Genome Sequencing

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By: Darci Butcher, PhD, and Rosanna Weksberg, MD, PhD

Genome sequencing initiatives  have been unable to identify the genetic causes or phenotypic modulators of many of disorders seen in clinical medicine. Layered on top of the DNA sequence is epigenetic information, defined as a “stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” (1). Identifying the epigenetic marks and characterizing how they are read to regulate the expression of the primary genomic sequence is necessary for our understanding of human development and disease. Multiple epigenetic mechanisms including DNA methylation at cytosine residues in CpG dinucleotides, covalent modifications of histone proteins, regulatory non-coding RNAs, including small interfering RNA (siRNA), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) participate in regulating gene expression and chromatin architecture. Disruption of these mechanisms is associated with a variety of diseases with behavioural, endocrine or neurologic manifestations and disorders of tissue growth, including cancer. The involvement of epigenetic alterations in many diseases has been known for some time, but only recently has it begun to be useful for clinical practice to diagnose and monitor disease progression.

In the Weksberg laboratory we determine genome-wide differential DNA methylation, gene expression and histone modifications for a number of disorders that have known or suspected aberrations in their epigenomic patterns. Many of these projects are collaborative efforts between the research laboratory and clinicians at the Hospital for Sick Children and around the world. We are identifying genes and pathways that have altered DNA methylation to determine their contribution to the overall disease phenotype. The projects in the laboratory can be separated into those related to growth, including genomic imprinting and intrauterine growth restriction and those related to neurodevelopment including autism spectrum disorders (ASD) and other paediatric neuropsychiatric disorders.

A number of disorders are caused by aberrant genomic imprinting resulting from unequal contributions of maternal and paternal alleles to the offspring2. Imprinted genes typically function in growth regulation and neurodevelopment, and the corresponding disease phenotypes are due to genetic or epigenetic aberrations in these genes often result in abnormalities of intrauterine growth or post-natal cognition and behavior. These disorders include Beckwith-Wiedemann (BWS), Silver-Russell (SRS), Prader-Willi (PWS) and Angelman syndromes (AS).

The molecular and epigenetic causes of Beckwith-Wiedemann syndrome (BWS) have been studied in depth in the Weksberg laboratory (3). This disorder is a rare, often sporadic, heterogeneous congenital overgrowth disorder which has many features including somatic overgrowth, large tongue, abdominal wall defects, ear creases and pits, kidney malformations and neonatal hypoglycemia, as well as in increased risk of embryonal tumours. BWS is caused by epigenomic and/or genomic alterations in the imprinted gene clusters on chromosome band 11p15.5 (4) can be subdivided into two distinct imprinted domains. Most cases of BWS are due to epigenetic lesions: either a gain of CpG methylation at an imprinting control region on the maternal allele of the H19 upstream differentially methylation region (DMR), which silences H19 and activates expression of the growth promoting gene insulin growth factor 2 (IGF2), or a loss of methylation at another imprinting control region of the KCNQ1intronic DMR, which silences the growth suppressor gene CDKN1C plus several nearby maternally expressed genes. Identifying specific molecular defects in imprinting disorders provides important information for patient management and for estimating recurrence risk. Molecular diagnostic testing for abnormal DNA methylation in the relevant imprinted domains can be done for a number of imprinting disorders and is already widely applied in PWS/AS and BWS/SRS. The majority of molecular alterations within imprinting domains can be identified by alterations in DNA methylation in the respective imprinting control regions. A few retrospective studies have shown an increased incidence of epigenetic abnormalities causing both BWS and AS following the use of assisted reproductive technologies (ART). Although the increased relative risk is small for these DNA methylation errors these data highlight the importance of understanding the mechanisms behind genomic imprinting.

The number of imprinted genes in human and mouse is currently just over 100 imprinted transcripts. Approximately 63 of these have been identified in humans. We designed experiments using uniparental tissues and DNA methylation at known imprinted genes to identify new imprinted genes (5). We took advantage of two uniparental tissues; complete androgenetic hydatidiform moles (CHMs) and mature cystic ovarian teratomas. CHMs have two copies of each paternal chromosome and no maternal chromosomes. Mature cystic teratomas, on the other hand, have two copies of the maternal genome. Analyzing the genome-wide DNA methylation patterns in these tissues and comparing them to normal biparental tissue we identified a number of candidate imprinted genes and validated one of those genes both mouse and humans (5). An expanded set of known imprinted genes could lead to the identification of the molecular cause of disorders of unknown etiology.

The laboratory is also interested in the epigenetic contribution to intrauterine growth restriction (IUGR), a heterogeneous disorder in which babies are born with a birthweight less than the 10th centile for gestational age. IUGR has been associated not only with significant maternal and fetal/neonatal mortality and morbidity but also with adult-onset disorders such as hypertension, coronary artery disease, and type 2 diabetes. DNA methylation alterations have been shown to drive increased or decreased placental and fetal growth. By determining DNA methylation patterns in the placenta of children born small for gestational age, we identified that gain of methylation in WNT2 was significantly associated with reduced WNT2 expression in placenta and with low birthweight percentile in the neonate (6). This gene has been demonstrated to have important function in mouse placental development. These data suggest that WNT2 expression can be epigenetically downregulated in the placenta by DNA methylation of its promoter and that high WNT2 promoter methylation is an epigenetic variant that is associated with reduced fetal growth potential (6). We expect that future studies of the epigenome will elucidate other candidate genes that undergo epigenetic dysregulation and negatively impact placental and fetal health.

The second focus in the Weksberg laboratory is the investigation of DNA methylation alterations in paediatric neurodevelopment and neuropsychiatric disorders. A number of neuropsychiatric disorders have been described with mutations or deletions in genes that are important for maintaining normal epigenetic regulation. Loss of function of these genes can disrupt normal establishment, maintenance, or reading of epigenetic marks, thereby resulting in altered chromatin structure and gene expression. In most disorders of this type, we still do not understand precisely how the mutation is related to the phenotype of the human disease. Many of these disorders are associated with intellectual disability (ID), as well as additional features including various congenital anomalies. The identification of alterations in DNA methylation associated with mutations in specific genes that function in epigenetic regulation will teach us more about what the responsibility of each epigenetic modifier is in the normal patterning of the epigenome. Other paediatric disorders we are investigating include autism spectrum (ASD), obsessive compulsive (OCD) and attention deficit hyperactivity (ADHD). For each of these disorders there have been genetic factors identified which explain a small proportion of such cases. We have proposed that epigenetic factors also contribute to the etiology of these disorders. These studies are all in the initial stages but we already have a number of interesting and encouraging results. We are currently investigating a number of candidate genes and pathways that may be relevant to these disorders.

The field of epigenetics is generating exciting discoveries in parallel to genome sequencing initiatives. The NIH Roadmap Epigenomics Mapping Consortium was launched to produce a public resource of human epigenomic data to catalyze basic biology and disease oriented research (7). Parallel initiatives include the NIH Epigenomics of Health and Disease Roadmap Program and CIHR Canadian Epigenomic Mapping Centres. These initiatives interface with the International Human Epigenomics Consortium, which was established to accelerate and coordinate epigenomics research worldwide (8). This is an exciting time for all researchers involved in epigenetic research as we work towards deciphering the language of the epigenome at an exponential rate.

Darci Butcher, PhD, Postdoctoral Fellow

Program in Genetics and Genome Biology,
The Hospital for Sick Children

Rosanna Weksberg, MD, PhD

Staff Physician, Clinical and Metabolic Genetics
Co-Director and Staff Geneticist, Cancer Genetics Program
The Hospital for Sick Children

Molecular and Medical Genetics
University of Toronto


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2.    Weksberg, R. Imprinted genes and human disease. American Journal of Medical Genetics Part C: Seminars in Medical Genetics 154C, 317-320.
3.    Choufani, S., Shuman, C. & Weksberg, R. Beckwith–Wiedemann syndrome. American Journal of Medical Genetics Part C: Seminars in Medical Genetics 154C, 343-354.
4.    Weksberg, R., Smith, A.C., Squire, J. & Sadowski, P. Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Human molecular genetics 12 Spec No 1, R61-8. (2003).
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8.    Eckhardt, F., Beck, S., Gut, I.G. & Berlin, K. Future potential of the Human Epigenome Project. Expert review of molecular diagnostics 4, 609-18 (2004).