lecture 26: epigenetics in human health and disease

Question 1

What is epigenetics?

Answer 1

• epigenetic modifications mediate changes in gene expression in the absence of change to underlying DNA sequence
• “the structural adaptation of chromosomal regions so as to register, signal, or perpetuate altered activity states” (Bird, 2007)
• chromosome-associated factors that regulate the activity of underlying DNA sequence
• cellular “memory”/’plastic’ in response to environment

Question 2

What are epigenetic modifications?

Answer 2

• epigenetic mechanisms are affected by these factors and processes:
  
  • development (in utero, childhood)
  • environmental chemicals
  • drugs/pharmaceuticals
  • ageing
  • diet
• health endpoints
  
  • cancer
  • autoimmune disease
  • mental disorders
  • diabetes
• DNA methylation
  
  • methyl group (an epigenetic factor found in some dietary sources) can tag DNA and activate or repress genes)
• Histone modification
  
  • the binding of epigenetic factors to histone “tails” alters the extent to which DNA is wrapped around histones and the availability of genes in the DNA to be activated
• variant histones

Question 3

What is the histone code?

Answer 3

• “writer” enzymes that catalyse modification
• “eraser” enzymes that remove modification
• euchromatin (open)
  
  • acetylation
  • maybe methylation
  • phosphorylation
• heterochromatin (closed)
  
  • lots of methylation

Question 4

What is DNA methylation?

Answer 4

• occurs primarily at CpG in vertebrates
• On/Off switch at gene promoters, dimmer at gene enhancers
• CpG island = dense region of CpG sites
• most CpG sites (greater than 90%) are dispersed around the genome at low densities
• methylated CpG site → blocked transcription
• unmethylated CpG site → transcriptionally competent

Question 5

In what way is chromatin a dynamic equilibrium?

Answer 5

• open or active euchromatic → closed or inactive heterochromatin
  
  • DNMTs, HDACs, HMTs, MBPs
  • adding hypermethylated histone tails
  • removing hyperacetylated histone tails
  • adding more methylated CpG
• other way
  
  • TETs, HATs, HDMs
  • RNA pol complex can access the gene and transcribe it

Question 6

What is X-chromosome inactivation?

Answer 6

• all female mammals silence one X chromosome
• expression of non-coding RNA (Xist)
• change in histone posttranslational modification
  
  • elevated H3K9, H3K27 methylation
  • loss of histone acetylation
• incorporation of variant histone protein
  
  • macroH2A
• association of chromatin modifying proteins
  
  • e.g. MBD, ATRX
• methylation of CpG islands in DNA

Question 7

What is spatial heterogeneity?

Answer 7

• 1 genome: 1000s of epigenomes
• sum total of epigenetic modifications within a cell
• every cell has a distinct epigenome
• (cumulative environmental factors)n →
• genotype →
• (cumulative stochastic influence)n →
• all lead to epigenotype → gene expression → phenotype

Question 8

Is DNA methylation static?

Answer 8

• no it is highly dynamic

Question 9

What is epigenetics in foetal programming and DOHaD?

Answer 9

• environmental exposure (e.g. diet)
  
  • sub optimal intrauterine environment
• stochastic factors
• genetic and sex specific effects
  
  • disruption in epigenetic profile
    
    • changes in gene expression
    • metabolic/endocrine disruption
      
      • modified tissue function/development
      • foetal programming/maladaption?
        
        • adverse birth outcome including low birth weight
          
          • predisposition to early life and adult onset disease (e.g. T2D)

Question 10

What are DNA methylation platforms?

Answer 10

• genome-wide DNA methylation analysis
  
  • infinium Human Methylation27 (HM27) and HM450 bead arrays
  • 27,000 CpG sites, 14,500 genes or 486,000 CpG sites - all genes
  • 12 samples/array (~$500/Sample)
  • 450 targets all regions
  • 27 targets promoter regions

Question 11

How variable is the early human epigenome?

Answer 11

• unsupervised clustering of 27,000 DNA methylation values (HM27) from human placenta across gestation
• watch this

Question 12

What is a heatmap of most variable probes (1st vs 3rd trimester)?

Answer 12

• increasing methylation

Question 13

What does the blood of premature infants show?

Answer 13

• large-scale epigenetic differences
• analysis of epigenetic changes in survivors of preterm birth reveals the effect of gestational age and evidence for a long term legacy

Question 14

What is DNA methylation and T-development?

Answer 14

• HM450 array analysis
• HT-12 expression analysis
• genome-scale profiling reveals a subset of genes regulated by DNA methylation that programme somatic T-cell phenotypes in humans

Question 15

summary thus far

Answer 15

• clear evidence of highly dynamic early life DNA methylation profile in multiple tissues, both before and after birth
• overall increase in methylation level and number of variable CpG sites over time in blood and placenta
• evidence for increasing drift (increasing variance) between individuals over time
  
  • supports a model of cumulative effects of environmental exposure on epigenetic profile during early life

Question 16

What is the relationship between epigenetics and the environment?

Answer 16

• the inherent sensitivity of epigenetic processes to subcellular environmental cues, that is a hallmark of differentiation and development, also render epigenetic profile sensitive to external environmental influence - Novakovic et al, 2013

Question 17

What is the influence postnatal maternal care?

Answer 17

• epigenetic programming by maternal behaviour
• low licking and grooming → stuff → decreased GR expression → high corticosterone levels, high anxiety, low licking or grooming
• high licking and grooming → increased GR expression → low corticosterone levels, low anxiety, high licking

Question 18

What are components of diet that can contribute a methyl group to DNA?

Answer 18

• folate
• vitamin B2
• vitamin B6
• vitamin B12
• choline
• all one carbon donors → SAM-e → primary methyl donor in all eukaryotes → addition of a methyl group

Question 19

What is the effect of maternal diet on the neonatal epigenome?

Answer 19

• a focus on folate
• maternal epigenetics and methyl supplements affect agouti gene expression in A/a mice

Question 20

What are other environmental epigenetic regulators as seen in animal studies?

Answer 20

• maternal care and stress
• endocrine disruptors
• folate and micronutrients
• assisted reproduction
  
  • superovulation and/or embryo culturing
• alcohol exposure
• smoking
• pollution/heavy metals/particulates
• many others emerging

Question 21

What is seen in a methylation analysis of twins?

Answer 21

• classical twin model
  
  • monozygotic (MZ) twins share 100% of genetic material
  • dizygotic (DZ) twins share ~50% of genetic variation
  • MZ correlation greater DZ correlation (genetic influence)
  • within pair MZ differences (environmental/stochastic influence)
• discordant monozygotic twins
  
  • insights into non-genetic mechanisms in disease

Question 22

What is the PETS timeline?

Answer 22

• 18-20 weeks - recruitment, diet, stress, lifestyle, conception questionnaires
• 28 weeks
  
  • maternal questionnaires (as above if needed)
  • maternal blood → serum/plasma storage
• birth - baby measurements, birth data, (questionnaire data)
  
  • cord blood → serum/plasma (-70C), WBC/CBMC (LN2)
  • placenta → multiple biopsies in RNA later (-70C)
  • cord tissue → biopsy (-70C), HUVECs (LN2)
  • buccal swabs → DNA (-70C)
• 18mth followup
  
  • questionnaires (diet, lifestyle, general), baby measurements
  • peripheral blood → serum/plasma (-70C)
  • buccal swabs → DNA (-70C)
• PETS = the peri/post-natal epigenetic twins study

Question 23

What is genome-wide methylation data?

Answer 23

• evidence of environmental effects
• clear evidence of variabilty within MZ twins

Question 24

What is the relationship between smoking and epigenetics?

Answer 24

• maternal
• DNA methylation of 1,062 newborn cord bloods from the Norwegian Mother and Child Cohort Study (MoBa)
• replication in newborn epigenetic study - NEST

Question 25

What is replication of methylation at 26 CpG sites?

Answer 25

Question 26

What are outstanding questions?

Answer 26

• how reproducible are the data in other cohorts?
• what is the size of any ‘regional’ effect?
• is there any tissue specificity?
• dosage and timing effects?
• functional relevance? is there an effect on expression?
• is there any evidence for stability postnatally – (epigenetic ‘legacy’) in the absence of continued exposure?
• do genetic factors contribute to inter-individual methylation variation?

Question 27

What is the regional effect of maternal smoking AHRR intron 1 methylation in CBMCs?

Answer 27

Question 28

Is there a difference between those who smoked early and those who smoked throughout pregnancy?

Answer 28

• yes - prolonged exposure is necessary

Question 29

What is the difference between buccal epithelial and placent of smoking vs non-smoking mothers?

Answer 29

Question 30

What is the difference in mean methylation between never smokers and smokers at birth and 18m?

Answer 30

Question 31

What role does genetics play in regulating the early life epigenetic profile?

Answer 31

• CBMCs
  
  • 11/16 MZ cluster - 69%
  • 5/10 DZ cluster - 50%
• HUVECs
  
  • 6/13 MZ cluster - 46%
  • 0/8 DZ cluster - 0%
• placenta
  
  • 7/8 MZ cluster - 88%
  • 3/6 DZ cluster - 50%
• contribution of genetic variation to transgenerational inheritance of DNA methylation
• the effect of geneotype and in utero environment on interindividual variation in neonate DNA methylomes
• GeMes, Clusters of DNA methylation under genetic control, can inform genetic and epigenetic analysis of disease

Question 32

Summary thus far

Answer 32

• variation in DNA methylation in MZ twins at birth highlights the importance of environment in specifying neonatal epigenetic profile
• clear evidence now exists confirming a genetic contribution to the human epigenetic profile
• known carcinogens (e.g. smoking) can induce stable epigenetic change even in utero

Question 33

What is evidence for epigenetic disruption in complex phenotypes/disease in humans?

Answer 33

• uniequivocal evidence for:
  
  • imprinting disorders (BWS, SRS)
  • ICF syndrome
  • all adult cancers
• evidence emerging for:
  
  • immune related (T1D, MS, atopy, asthma, arthritis)
  • neurological (bipolar, schiz, MD< eating disorders, Alzheimers, Parkinsons)
  • musculoskeletal (osteoporosis)
  • metabolic (type II diabetes, obesity)
  • cardiovascular (foetal programming)

Question 34

What is epigenetic disruption in complex human disease?

Answer 34

• personalised epigenomic signatures that are stable over time and cobary
• genome wide survey reveals predisposing diabetes type 2-related DNA methylation variations in human peripheral blood

Question 35

Is there replication of T2D findings?

Answer 35

• genome-wide DNA methylation analysis of human pancreatic islets from Type 2 Diabetic and Non-Diabetic donors identifies candidate genes that influence insulin secretion
• epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy
• genome-scale case control analysis of CD4+ T cell DNA methylation in juvenile idiopathic arthritis reveals potential targets involved in disease

Question 36

What kind of disease is cancer?

Answer 36

• epigenetic
• CpG island methylation → methylation spreading
• tumour suppressor gene expression potential → gene silencing
• widespread hypomethylation (for example at late-replicating LADs)
  
  • mislocalisation of DNMT1

Question 37

What is utilising archived clinical material for DNA methylation analysis?

Answer 37

• bone marrow is taken for patient diagnosis
• excess bone marrow is archived
• suitable for methylation analysis (Wong, 2008)
• retrospective cohort of more than 600 patients with clinically annotated samples, including outcome

Question 38

What defines paediatric pre-B cell acute lymphoblastic leukaemia?

Answer 38

• a distinct DNA methylation signature
• genome wide analysis of 17 matched pairs of ETV6-RUNX1 subtype paediatric ALL cases (infinium 27k methylation microarray - targeting gene promoters)
• heatmap shows 115 probes with leukaemic specific methylation
• associated with genes previously implicated in leukaemia, other malignancies and haematopoietic development
• A 15 gene signature accurately defines ALL
  
  • validated on a subset of 85 mixed ALL patients
• represents a pan-ALL biomarkers
• the diagnostic potential of a subset of the markers was investigated

Question 39

What is DNA methylation as a biomarker for disease?

Answer 39

• watch this

Question 40

What is epigenetic deregulation in paediatric acute lymphoblastic leukaemia?

Answer 40

• watch this

Question 41

What are prognostic signatures in paediatric pre B-cell ALL?

Answer 41

• LAT1 intra-genic DNA methylation is associated with relapsed paediatric ALL

Question 42

What is other recent cancer research?

Answer 42

• stability of gene expression and epigenetic profiles highlights the utility of patient-derived paediatric acute lymphoblastic leukaemia xenografts for investigating molecular mechanisms of drug resistance
• integrated genomic analysis of relapsed childhood acute lymphoblastic leukaemia reveals therapeutic strategies
• hypermethylation and down-regulation of DLEY2 in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a-16-1
• optimised DNA extraction for methylome profiling using neonatal dried blood

Question 43

summary 3

Answer 43

• mounting evidence links distinct epigenetic change to complex phenotypes in humans
• in some instances evidence of epigenetic change exists prior to phenotypic onset
• all human cancers show a disrupted epigenetic profile
• epigenetic profiling has utility at multiple levels in paediatric cancers
  
  • diagnosis, disease monitoring, prognostication

Question 44

What are caveats?

Answer 44

• general lack of reproduction
  
  • differences in phenotyping
  • different analytical approaches
• generally insufficient sample size
  
  • technological and $$$ limitations
• general lack of assessment in appropriate target tissue
• lack of longitudinal analysis - cause vs effect?
• efect sizes are often questionable

Question 45

What is the relationship between methylation and alzheimer’s?

Answer 45

• methylomic profiling implicates cortical deregulation of ANK1 in Alzheimer’s disease
• Alzheimer’s disease: early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci

Question 46

What is the prevailing model?

Answer 46

• epigenetic variation, disease and ageing
• determined by:
  
  • genetic variation
  • environment/lifestyle (including diet)
  • stochastic factors
  • telomere length
  • other?

Question 47

What are conclusions?

Answer 47

• epigenetic variation defines who we are
• mounting evidence links environmentally induced epigenetic change in utero to altered phenotype in animal models
• the in utero period is critical in determining the overall epigenome in humans
• the early life epigenome is highly dynamic and sensitive to environmental influence
• also regulated by underlying genetic effects in a tissue specific manner
• preliminary data link altered DNA methylation to complex diseases such as allergy and cancer in children AND to specific exposures in utero