11/10/2017
Pain Genetics and Epigenetics
Stephen McMahon King’s College London London Pain Consortium
Disclosures
Research support 1) Via EU-IMI support of ‘Europain’: Astra Zeneca, Lundbeck, Pfizer, Lilly, Boehringer Ingelheim, Esteve, SanofiAventis, Grünenthal, AbbVie, Astellas, Neuroscience Technologies 2) Nevro Corporation 3) Convergence Pharmaceuticals Ltd & Evotec. 4) GSK.
Studentships 1) CASE studentship with Medimmune 2) PhD studentship with Pfizer
Consultancies Pfizer Bayer healthcare Astra Zeneca Mundipharma Glenmark Pharmaceuticals Regeneron Lundbeck Afferent Pharma
Why does tissue injury cause pain?
Rene Descartes 1596-1640
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We have a good understanding of why injury cause pain
TRANSDUCTION MODULATION
Heat Cold Damage mediators Glutamate ATP TRPV1 TRPM8 P2X3 P2Y Neuropeptides BDNF TRPV2 TRPA1 Bradykinin B1/2 Inhibitory controls TRPV3 5-HT3 and other 5HT MOR CB1/2 GABA TRPV4 Mechanosensation Microglial Activation P2X3 TRPV4 ATP via P2X4,P2X7 TRPC/V/P? CSF1, CCL2, BDNF, Inflammatory mediators & targets Fractalkine Prostaglandins, NGF, IL-1, TNF etc. Ligand & Voltage-gated channels e.g. TRPV1,Nav1.9, Nav1.7, Nav1.8
TRANSMISSION
Nav1.7, Nav1.8 Nav1.1, Nav1.2, Nav1.6 (Nav1.3) Threshold - Nav1.9
Why is the pain experience so variable?
Experimental pain in healthy human subjects varies enormously 50 C) o 48
46
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42 Mean +/- 2 x std dev
Heat Pain Tolerance ( Tolerance Pain Heat 40 >2000 twins tested 35 40 45 50 Heat Pain Threshold ( oC)
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Pain and pathology are only weakly correlated
Kellgren-Lawrence scores of radiographic damage Prevalence of pain 70 60 50 40 Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 30 Magnitude of pain 20 100
Arendt-Neilsen et al., 2014 %patientswith pain 80 N=281 10 N=10,278 60 N 0 40 K/L 0/1 2 3/4 20 ♂carpal ♂wrist ♂hand 0 ♀cervical ♂lumbar ♂hip
Pain in last in Pain 24hlast (VAS) 0 1 2 3 4 foot Lawrence et al., 1966 Kellgren/Lawrence score ♀
Pain can persist when pathology resolves
* 10 50 Chronic Widespread pain 8 34% Chronic Regional pain 40 * * * * No chronic pain 6 30 46% mean VAS meanVAS
Swollen joints 4 * 20 * 20% 2
10 24 60 Ent. 6 12 24 60 months Ent. 6 12
1,386 RA patients followed longitudinally Andersson et al., 2013 J Rheumatology
What explains this variability?
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What explains pain variability? Genetic factors Single gene mutations can result in abnormal pain states, but are very rare Phenotypes are dramatic and don’t explain pain normal variability Loss of function Gain of function
Courtesy Dave Bennett Normal HSN IV Sural nerve patient
Known genes: NGF/TrkA; Na v1.7; Known genes: Na v1.7; Na v1.8; PRDM12 Na v1.9; TrpA1
Genetic influences on Pain – MONOZYGOTIC DIZYGOTIC Twin Studies
• Heat Pain Threshold (HPT) • Pain during creation of 45 °C thermal burn • Primary & secondary hyperalgesia after burn symptom-based classification 30 • Pain during iontophoresis of Acid + ATP Flare 25 Static Allodynia • Itch after iontophoresis of Histamine Dynamic 20 Allodynia 15
Area (cm2) Area 10 n = 20 5
0 0 5 10 15 20 30 40 50 60 Time (mins) 45 oC for 330sec Tim Norbury
Heritability of pain traits – twin studies
Shared Monozygotes Modality Rmz Rdz Heritability Environment 52 Twin2 HPT (°C) 0.62 0.26 0.57 50 Pre-Burn HPT 0 48 (0.46-0.79) (0.01-0.52) (0.40-0.70) 0.45 46 Post-Burn HPT 0.49 0.28 0 (0.29-0.69) (0.03-0.54) (0.26-0.60) 44 Pain Rating during 0.30 0.08 0.30 42 0 n=110 burn creation (0.06-0.54) (0.00-0.35) (0.05-0.51) 40 R=0.6 Pain during ATP 0.27 0.00 0.24 38 2 0 38 40 42 44 46 48 50 52 iontophoresis (0.02-0.52) (0.00-0.29) (0.00-0.45) Pain during acid 0.28 0.13 0.27 Twin 1 HPT (°C) 0 iontophoresis (0.03-0.52) (0.00-0.41) (0.04-0.47) Dizygotes Itch after histamine 0.19 0.34 0.2 0
Twin2 HPT (°C) 52 iontophoresis (0.12-0.62) (0.00-0.35) (0.09-0.52) 50 2°pinprick 0.60 0.29 0.59 0 48 hyperalgesia area (0.43-0.77) (0.04-0.54) (0.41-0.73) 46 2°brush evoked 0.35 0.55 0.38 0 44 allodynia area (0.36-0.74) (0.19-0.57) (0.00-0.74) 42 n=102 0.76 0.56 0.69 2°skin flare area 0 40 R=0.26 (0.66-0.87) (0.38-.075) (0.57-0.78) 38 38 40 42 44 46 48 50 52 Twin 1 HPT (°C) Norbury et al., 2007
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Heritability of pain traits from twin studies Authors Subject details Cohort Heritability Phenotype examined MacGregor et al., Female twins Twins UK 0.52-0.68 Lower back pain 2004 (181 MZ, 351 DZ) 0.35-0.58 Neck pain Norbury et al., Female twins Twins UK 0.22-0.59 Quantitative sensory 2007 (51 MZ, 47 DZ) testing Battie et al., 2007 Male twins Finnish twin cohort 0.3-0.46 Lower back pain (147 MZ, 153 DZ) Nielsen et al., 2008 Mixed twins Norwegian twin 0.6 Cold-pressor pain (53 MZ, 39 DZ) registry 0.26 Heat contact pain Altman et al., 2011 Female twins Swedish twin 0.3 Bladder pain syndrome (1,867 MZ, 1,293 DZ) registry Williams et al., 2010 Mixed twins Twins UK 0.46 Pain at different body sites (991 MZ, 1,074 DZ) Markkula et al., Mixed twins Finnish twin cohort 0.51 fibromyalgia 2009 (12,500) Kato, Sullivan and Mixed twins Swedish twin ♂0.09 Chronic widespread pain Pedersen, 2010 (28,531 pairs) registry ♀0.13 Hocking et al., 2012 2,195 extended Scottish family 0.16 Any chronic pain families health study 0.3 Severe chronic pain Livshits et al., 2011 Female twins Twins UK 0.43-0.68 Lower back pain (371 MZ, 698 DZ) Hartvigsen et al., Mixed twins Danish twin registry 0.33-0.39 Back pain (lumbar, 2009 (6,700 MZ, 8,500 DZ) thoracic and neck) Polderman et al., Mixed twins Meta-analysis 0.42 Migraine 2015 (9,019) (0.36-0.47) Denk 2015
What genes contribute to pain heritability? GWAS – comparing the genomes of different individuals
1 2 3
SNP1 SNP2 Case Con Case Con
Ask if a particular SNP is associated with a given trait. Significance Human genome ~ 3billion base pairs. SNPs on average every 300 nucleotides. Any 2 individuals likely to Test the statistical significance of the differ by <0.5%. association. Does not imply disease causing.
GWAS in large numbers of migraineurs identifies 44 SNPs Gormley et al. 2016, Nat Genetics
OR= 1.111.11 0.941.06 1.09 1.11 0.90 0.89 1.07 -log value P -log
Chromosome
60,000 migraineurs, 316,000 controls Odds Ratio of individual loci typically 5-10% Only 2 loci close to neuronal channels Most SNPs are in regulatory regions 6 associated with NO signalling Together only explain 10-20% of the heritability 30% associated with vascular function
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Do rare variants explain a lot of the variability? 50 C) o 48
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Heat Pain Tolerance ( Tolerance Pain Heat 40
35 40 45 50 Heat Pain Threshold ( oC) Williams et al., PLoS genet, 2012
Rare variants in painful diabetic peripheral neuropathy: candidate gene approach (Nav1.7) Iulia Blesneac/Dave Bennett lab
190 Diabetic Peripheral Neuropathy ‹ Nav1.7 published variants (5) ‹ Nav1.7 unpublished variants (6) 11 0 WT M1852T T1596I PAINLESS 79 PAINFUL 111
S802G T1596I
I II III IV
K655R K1043N M1852T I564T
Pain sensitivity varies enormously in health and disease
Monogenic Monogenic
NGF/TrkA Nav 1.7 Nav 1.7 Nav 1.8 (N av 1.9) Nav 1.9 PRDM12 TrpA1 Frequency
Insensitive Normal Hypersensitive Polygenic/epigenetic Pain sensitivity
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Beyond genetics: Meta-analysis of Post-operative pain . Gerbershagen et al., 2014 Pre-op Pain Age Sex ♀ Environmental risk factors Knee Hernia Gastr. Hyster. Pain ‘history’ Append. Shoulder Hip (experimental pain Hyster. Chole. thresholds, prior pain) Spine Nasal Lamin. Psychosocial factors Hernia Thyroid (expectation, catastrophising, Hernia Fistula depression, anxiety) Mast. Hernia Hernia Age Sinus BCT BCT Gender Sling Veins Early life stressors Prostate Bladder Total ~23,000 0.9 1.40.4 1.2 0.5 3.0 Odds ratio to develop pain
Mechanisms of acquired vulnerability: altered brain connectivity
Mechanisms of acquired vulnerability: Epigenetics
Cells have the same genes. Different genes are switched on or off in different types of cell. These regulatory patterns traditionally considered stable in maturity.
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Genetics Epigenetics
PTMs Histone Variants Mutations DNA methylation
Permanent Phenotypic inheritance Plastic ‰ Stable Species maintenance Memory of transitional change Somatic variability and development Response to environmental cues
DNA methylation
DNMTs guanin
e CH 3
cytosineTet CpG island
Transcriptional start site
Methylated CpG Unmethylated CpG Typical Gene http://www.roadmapepigenomics.org/overview
Histone modifications.
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Multiple proteins participate in chromatin modifications.
Epigenetics: relevance to pain
1. Potential for novel therapeutic targets
2. Pain chronification & vulnerability
Many genes are dysregulated in chronic pain states
L4 DRG, 14 day spinal nerve injury
Down Up 1012 1198 adjvalue P 10 Log
HDAC1 HDAC4
Log 2 Fold Change Ana Antunes-Martin
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HDAC4 cKO mice have normal acute pain responses
Von Frey Hargreaves Tail Flick 0.8 9 18 8 16 0.6 7 wt Nav1.8 cKO 14 6 fl/fl 12 5 Hdac4 0.4 10 4 Hdac4 Nav1.8 HDAC4 8 3 6 0.2 2 4 α-tubulin 1 2 0 50% threshold (g) 50% threshold 0 0 Withdrawal latencyWithdrawal (s) 49°C 52°C Withdrawal latency (s) latency Withdrawal 0.6 Adv 20 cKO fl/fl wt 0.5 18 9 Hdac4 16 8 Hdac4 Adv HDAC4 0.4 14 7 12 6 0.3 10 5 4 α-tubulin 8 0.2 6 3 4 2 0.1 2 1 50% threshold (g) threshold50%
Withdrawl latency (s) 0 0
0 Wtihdrawal (s) latency 49C 52C Crow et al., FASEB 2015
HDAC4 cKO mice have reduced inflammatory pain
fl/fl cKO HDAC4Nav1.8 CFA ipsi HDAC4Adv CFA ipsi 12 7 Baseline 10 Baseline 6
8 5 4 6 3 4 HDAC4 fl/fl 2 HDAC4 fl/fl Withdrawal (s) Withdrawal latency
Withdrawal (s) Withdrawal latency 2 1 HDAC4 Nav1.8 cKO HDAC4 Adv cKO 0 0 Day 3 Day 7Day 10Day 14 Day 3 Day 7 Day 10Day 14
Crow et al., FASEB, 2015
Epigenetics: relevance to pain
1. Potential for novel therapeutic targets
2. Pain chronification & vulnerability
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Activated glia contribute to some neuropathic pain states.
IBA1 immunostaining
40 Saline IL-1β, TNF α, IL-6 Minocycline NO, PGs, BDNF 35 30
Marchand et al., Nature Neuroscience Reviews 2005. 25 Beggs et al., 2012, 20 Nat Neurosci. Pain threshold (g) Painthreshold 15 Con Neuropathic
Persistent pain states are associated with altered epigenetic signatures
Sham Regions enriched for H3K4me1 = enhancers
Neuropathic
novel primed enhancer
CCL12 In experimental neuropathy new enhancers emerge in the regulatory regions of some genes in spinal cord cells Denk et al. Cell Reports, 2016
Novel enhancers day 7 Dysregulated transcripts day 7
SNL SHAM Closest TSS SHAM SNL Fam65b C4b Tmem176 Ccl12 Kif2c Fcgr2b Trim69 Ly86 120 Kif2c Eef1a1 Ccr2 13 45 Ccr3 Dusp12 8 Ptpn22 3 0 Defb48 normalised read countGm4759 log2(counts) MACS1.4 followed by diffBind SNL vs. SHAM, n = 4, deseq2, package at FDR < 0.1 log10(FPKM), adj. p < 0.05; Enhancers remain when transcription reverts to normal Denk et al., 2106
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Methylation studies in heat-pain discordant monozygotic twins (Jordana Bell)
CpG island 50
Transcriptional Methyl group
start site C) 48 o
46 Typical Gene
44 Phenotype ~2500 twins for thermal pain sensitivity 42 Identify 25 pairs of MZ twins with biggest
difference in noxious heat sensitivity ( Tolerance Pain Heat 40
Genome-wide sequencing after methylated DNA 35 40 45 50 Replication cohort of 50 unrelated individuals Heat Pain Threshold ( oC)
Differential methylation of DNA is associated with pain at various loci Heat Pain tolerance (oC) tolerance Pain Heat
TRPA1 MeDIP signal C) o
FirstFirst example example of anof epigenetican epigenetic ‘mark’ ‘mark’ associated associated with altered with pain ( tolerance Pain Heat sensitivity in humansaltered pain sensitivity Bell et al. Nature Comms, 2014 TRPA1mRNA expression
Pubmed finds 145 articles on “epigenetics AND pain”. 66 are reviews
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Pain vulnerability: implications for personalised medicine
Vulnerability/ = Genetic factors + Environmental factors Resilience 40% 60%
Individual genetic variants Twins studies recognize shared and contribute very little risk unique environmental factors
Together, genetic variants explain Surprisingly, most twin studies find only a fraction of known heritability that shared environmental factors predict very little personal risk If gene x gene interactions are important, they will be very difficult If gene x environment interactions to identify are important, they will be very difficult to identify
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