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Symptom/Metabolome-Directed Genomics of ME/CFS by Dr Neil McGregor (2017)

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Neil McGregor's presentation at Open Medicine Foundation's Community Symposium, "Molecular Basis of ME/CFS". Stanford University, August 12, 2017.

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Symptom/Metabolome-Directed Genomics of ME/CFS by Dr Neil McGregor (2017)

  1. 1. SYMPTOM/METABOLOME DIRECTED GENOMICS FOR ME/CFS NEIL R MCGREGOR PHD, CHRISTOPHER W ARMSTRONG BSC, PAUL GOOLEY PHD, HENRY BUTT PHD, BIO21 INSTITUTE, UNIVERSITY OF MELBOURNE, AUSTRALIA. JOHN WHITING MBBS, BCH, BAO, FRACP, SPECIALIST INFECTIOUS DISEASES AND INTERNAL MEDICINE, BRISBANE, AUSTRALIA DON LEWIS MBBS, CFS DISCOVERY, DONCASTER VICTORIA, AUSTRALIA.
  2. 2. ME/CFS IS A HETEROGENEOUS SYNDROME • Multiple studies have failed to find a single metabolic anomaly for ME/CFS. • Multiple different definitions of ME/CFS have been assessed also without success. • ME/CFS can only be concluded to be a heterogenous cluster of genetic/epigenetic events resulting in a common set of host responses which form part of the definitions. • Patients with similar onsets have different long term outcomes!!! Why??? • Patterns or clusters of sub-symptoms can be identified. • Regression modelling of the symptoms/ metabolomes shows that: • FATIGUE is best predicted by a change in glucose: a form of insulin resistance (Armstrong et al 2015). • WIDE SPREAD PAIN is best predicted by changes in kidney handling of electrolytes and amino acids (McGregor et al 2016). • Each of these symptom clusters must have an underlying genetic/epigenetic origin. Our approach is to use Symptome/Metabolome directed assessments.
  3. 3. GENOME WIDE PILOT SNP STUDY DATA • 56 CFS patients and 32 controls. 553,000 SNPs assessed of the 610,000 available. Excluded those with multiple misreads, and uniform insertions and deletions. • All patients had MECFS for >3-years and had not recovered. • 316 SNPs were statistically significant with a z-score of ≥6.5. • 162 (51%) SNPs were from 118 identifiable proteins/enzymes (Virtually all no coding regions). • 49 (16%) SNPs were from 42 RNA genes. • 142 (33%) were from intergenic sections of DNA without any known ability to produce a protein but may be promoters or enhancers of other genes. • To eliminate control sample aberrant distribution the data cross checked against 1000 genomes (Phase 3 May 2013). • After 1000 genome cross check only 111 SNPs remained. Removed all with an odds ratio <2. Only 38 genes remained. • A dataset was derived for all the SNPs associated with the 38 genes and any other gene that overlapped the gene which may influence it expression.
  4. 4. GENETIC SUB CLUSTER ANALYSIS 1. Anomaly in G-protein coupled receptor protein. 2. Anomaly in G-protein coupled receptor protein and a STAT3 inflammation inhibitor gene. 3. Anomaly in G-protein coupled receptor protein and RNA helicase. 4. Anomaly in RNA helicase and Interleukin L-1 Kinase 3. 5. Anomaly in RNA Helicase and a viral adherence receptor. 6. Anomaly in RNA Helicase and Hedgehog Signalling. 7. Anomaly in RNA Helicase and Langerin (Dendritic cell pathogen receptor).
  5. 5. ODDS RATIOS OF MAJOR SNPS INCREASED IN ME/CFS Gene SNP Heterozygote OR (95%CL) p Homozygote OR (95%CL) p G-protein SNPs SNP a SNP b 5.4 (1.8-16.3) <.002 4.7 (1.6-14.1) <.005 NS NS RNA helicase SNPs SNP a SNP b 6.7 (2.4-18.4) <.0002 6.7 (2.4-18.4) <.0002 NS NS Langerin SNPs SNP a SNP b 3.6 (1.4-9.5) <.008 NS 6.7 (1.8-25) <.004 9.0 (1.9-42)<.005
  6. 6. DISTRIBUTION OF GENE SNPS: C V MECFS 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Control MECFS 2+ 0.06 0.48 1 0.52 0.33 0 0.42 0.19 %Subjects Langerin SNPs 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Control MECFS 9+ 0 0.13 5-8 0.22 0.4 1-4 0.47 0.45 0 0.31 0.02 %Subjects G-Protein SNPs 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Control MECFS 4 0.14 0.32 3 0.28 0.32 2 0.24 0.26 1 0.1 0.1 0 0.24 0 %Subjects RNA Helicase SNPs Carriage of a mutated SNP within the ME/CFS and the controls. Odds Ratios: 3.0 (95%CL 1.1-8.3) p<.02 21.5 (95%CL 2.5-185) p<.0002 12.6 (95%CL 3.5-45.0) p<.00001. .
  7. 7. G-PROTEIN COUPLED PROTEINS. • Heterotrimeric receptor GTPase (G-Protein Coupled Receptors) • 3 Subunits • Alpha • Beta • Gamma • The Alpha protein is highly mobile. • The Beta and Gamma chains are tightly bound and function as one. • There are 20 alpha, 6 beta and 13 gamma proteins. • Different combinations have different intracellular actions.
  8. 8. G-PROTEIN COUPLED RECEPTOR (GPCR) PATHWAYS • Activation of the receptor binds GTP to the alpha protein. • The GTP-α and GTP-βγ proteins separate and perform different functions.
  9. 9. GPCR RECEPTOR FUNCTIONS AND ME/CFS PATIENTS Symptoms influenced by receptor functions 1. Gustatory signalling (Taste reactivity). 2. Olfactory signalling (Smell reactivity). 3. Behaviour/Mood regulation (Serotonin, Dopamine, GIRK receptors). 4. Inflammation related (Histamine, NF-Kappa mediated transmission). 5. Parasympathetic and Sympathetic responses (Heart rate, blood pressure). 6. Homeostasis modulation (Water balance, electrolytes). 7. Visual sensing: translation of electromagnetic radiation (Rod and Cone light signalling, Photophobia). ME/CFS % 1. 40-50% 2. 40-50% 3. 50+% 4. 90+% 5. 70+% 6. 50+% 7. 50+%
  10. 10. RNA HELICASES • Cytoplasmic viral RNA sensing proteins. • These bind to the HMGB proteins and remove viral RNA from the cytoplasm. • Inhibited by Epidermal Growth Factor activation. • EGF receptor activated by Estrogen receptor beta and by some viruses. • Required for interferon production. RH 1 RH2 RH3 RH1
  11. 11. CD207 (LANGERIN) • CD207 (AKA Langerin) is a protein largely expressed in Dendritic (Langerhan) cells in skin, the GUT and multilayered epithelium. • Considered the first line of defense for viral and non-commensal bacteria infections. • It is a lectin detecting protein and is involved in detection of multiple viruses and in presentation of antigens from these pathogens to t-cells and the immune system.
  12. 12. LANGERIN AND HIV (VAN DER VLLIST AND GEIJTENBEEK 2010) Normal response. Viral degradation and presentation of antigens to the t-cell. Inhibition of Langerin. HIV binds to CD4 receptor and invades the host.
  13. 13. LANGERIN AND VIRUSES • Multiple viruses are able enter the cell through a Langerin mediated process. • Most of these viruses are associated with exacerbation of the illness in MECFS patients. Virus Reference HIV De Jong et al 2009 HSV1 De Jong et al 2010 HSV2 Puttur et al 2010 EBV Braz-Silva et al 2011 CMV Huang et al 2012 Influenza GeurtsvanKessel et al 2008 Measles Van der V et al 2011 HPV Bousarghin et al 2005 Dengue Limon-Flores et al 2005
  14. 14. MITOCHONDRIAL DNA. • Only one mtDNA SNP was increased in MECFS. T456C (C=0% v ME=18%; p<.02). • Located in the hypervariable region it is a defining SNP for Haplogroup H5. • Assessment of the autosomal genes carried by the H5 patients revealed they all carried the CD207 (Langerin) SNP mutations. • We found no evidence of a mitochondrial DNA anomaly.
  15. 15. SUMMARY • Using the symptom/ metabolome directed approach appears to have allowed us to delineated up to 7 underlying genetic cluster issues in the ME/CFS patient group. • Anomalies in G-Protein coupled receptor proteins activity may be related to the increased prevalence of orthostatic hypotension, photophobia, smell and taste hypersensitivity. • Anomalies in RNA Helicases may be related to the Viral infection, viral induced onset and viral reactivation events in MECFS patients. • Anomalies in Langerin may relate to the increased pathogen related issues. • No evidence of a mitochondrial genetic disorder.
  16. 16. • We need to very wary at this point in time. • We need to be sure what we have found DOES MATTER and is not DOESN’T MATTER. • Members of the conference scientists aim to do this confirmation. www.melbournebioanalytics.org

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