Genetics. Unravelling The Mysteries Of Methylation & Part 2. Recapping From Last Time. Forms Of Folate. September Folic Acid Is Not Folate!

Transcription

1 Unravelling The Mysteries Of Methylation & Genetics Part 2 Warren Maginn BHSc (Nutr. Med.) GradCert (Hum. Nutr.) for 1 Recapping From Last Time 214 Forms Of Folate Folic Acid Is Not Folate! Folic acid: Synthetic Very Stable Less readily metabolised more prone to bio accumulation (Accumulation can obstruct other folate intermediates/actions) Folates (DHF, Folinic, 5MTHF): Naturally Formed Relatively Unstable (e.g. cooking) Relatively easier to metabolise Metabolic blocks still occur (Due to genetics, toxicity, nutr. def..) 3 1

2 Under & Over Methylation: An Over Simplification? Body regulates methylation up and down locally (according to needs and risks) Upregulation is restricted by limits of capacity We need to support capacity where it is insufficient BUT not overwhelm where it is beneficially downregulated Balance Is Key How Do We Know What To Do? 4 Clinical Testing (How To Assess) 5 Testing Issues Standard Serum/Red Cell Folate testing: Does not show break down of multiple Folate forms (Can mask excess accumulation/metabolic dysfunction) Can therefore give false negatives (of functional deficiency) Does not necessarily assess levels in the CNS Testing Homocysteine alone: Homocysteine levels subject to innumerable factors Cannot distinguish between poor methylation or sulfation (Can give false negatives if TransSulfation is upregulated) i.e. Hard to draw approp. therapeutic conclusions from Hcys alone 6 2

3 Testing Issues Standard Serum B12 and FBC Testing: Standard hemotological signs of B12 deficiency can be masked by synthetic folic acid fortification/supplementation Direct serum Vitamin B12 measurements notoriously unreliable (poor approximation and does not show functional need) 2014 distinguishing subjects with normal and cobalamin deficient conditions by a threshold level of serum B12 concentrations is unreliable and clinically misleading 7 Where To From There? 8 Performing Genetic Tests OR 9 3

4 OR 10 How Are SNPs Reported By Genetic Tests? 11 Describing The Right SNP For Substitutions 1. Gene is identified (by acronym) e.g. MTHFR 2. SNP location is identified (by number) e.g Common variation type specified BEFORE Rarer variant type is specified AFTER Methods of Describing Gene Base Pair Sequence Amino Sequence Rs No. MTHFR C667T 667 C>T Ala222Val A222V rs For Insertions/Deletions SNPs reported simply as Present or Absent 12 4

5 SNP Test Results Once a SNP has been identified and analysed (e.g. C667T) The status of it s variation is reported as either: Status Description Methods Of Reporting Normal Wildtype / CC (C667T) Heterozygous Heterozygote /+ CT (C667T) Homozygous Homozygote +/+ TT (C667T) CC667 C667T 677TT 13 Proposed Criteria Before asnp Is Included in Clinical Analysis 1. Relevance SNP exerts a direct influence on specific biochemical processes that create known symptoms or disease 2. Prevalence Relatively common in the general population 3. Modifiability Expression of SNP is modifiable by known nutrition, environment or lifestyle interventions 4. Measurability Impact of clinical interventions on SNP expression is measureable by lab biochemistry 14 Genetic Testing Pitfall The impact of false/negative determinism can negate the potential usefulness/benefit of testing! i.e. The presentation/interpretation of gene results should not be inflammatory or negative 15 5

6 Gene Testing Unscrupulous Mail Order Laboratories Prey On Fears 16 Traffic Light Methods Impartial Method 17 Key Methylation SNPs 18 6

7 MTHFR Methylenetetrahydrofolate Reductase 19 Many SNPs Affect Methylation, But Which Ones Are Relevant?

8 677 C>T C swapped to a T at position 677 on the MTHFR gene One of most common SNPs affecting human health Reduces the MTHFR enzyme stability/activity by up to: 35% (65% activity) Heterozygous 75% (25% activity) Homozygous T Alleles yield: MTHFR enzyme activity = Homocysteine levels in DNA Methylation = in DNA adducts (reduced repair) Thus, associated with increased risk for: Cancer, CVD, NTDs, miscarriage, nephropathy, schizophrenia Means: Greater than normal Folate & B vitamin requirements 22 How Common is C677T? European African Mixed 23 Redefining ADEQUATE In 1999 we obviously hadn't completed our investigation When folate status is adequate, plasma homocysteine levels are normal and independent of genotype Bailey, Recent Adv Nutr Sci, 1999?? 24 8

9 TT genotype = higher Hcys and lower serum Folate Shelnutt K, et. al J. Nutr. 25 Which Form To Supplement? (To Restore Levels) 5MTHF FOLIC Willems et al. (2004) 26 When plasma Folate is high enough, Hcys can be normalised in the TT genotype 27 9

10 1298 A>C A swapped to a C at position 1298 on the MTHFR gene Affects MTHFR enzyme regulation with SAMe Unlike position 677, does not increase Hcys Assists BH4 metabolism (Neurotransmitters & Ammonia) Tends to affect Nervous System more than CVS (May decrease Catecholamines: Serotonin, Melatonin, Dopamine, Norepinephrine, and Epinephrine) AA = 0% loss of function AC = 20% loss of function or 30%? CC = 40% loss of function or 70%? 28 Compound Heterozygotes When more than one Heterozygous (+/ ) SNP is found within the same gene e.g. MTHFR 677 C>T CT (Hetero) MTHFR 1298 A>C AC (Hetero) Gene Specific Location Within Gene Common Vs Rare Variant Actual Result 29 Sample Test Result 30 10

11 DHFR Dihydrofolate Reductase 31 DHFR Dihydrofolate Reductase Current results have been inconsistent An example of a SNP that is best NOT included in clinical workup until more research can be done However illustrates the breadth of variability in Folate metabolism Illustrates need for optimised supplementation regimes (rather than indiscriminate forms and doses by either blanket fortification or untailored clinical interventions) 32 CBS Cystathionine Beta Synthase 33 11

12 CBS Cystathionine Beta Synthase 699 C>T (and 1080 T>C) CBS catalyses Hcys to Cystathionine (removal from Met. Cycle) T allele may increase activity? (SNP unlikely to directly alter enzyme but effect suspected via other unknown functional SNP) Significantly less CVD seen in TT (5% vs 17%) Almost 3x Hcys lowering response to Folate supplementation B6 may potentiate (without necessarily supporting methylation) Increased Taurine and Ammonia may yield sulphur intolerance May increase Vitamin B6 needs (to ensure maintained transulfation i.e. detox/antiox) 34 Sample Test Result 35 MTR Methionine Tetrahydrofolate Reductase 36 12

13 MTR Methionine Tetrahydrofolate Reductase (Methionine Synthase) 2756 A>G MS catalyses the remethylation of Hcys to Methionine G allele increases activity of the enzyme Enhancing conversion of homocysteine to methionine Thus Homocysteine tends to be lower with this SNP Dose dependant decline across genotypes (AA > AG > GG) Summary: a potentially beneficial variant (3.4x reduction in CVD) However may increase requirements for B12 (possible depletion) 37 Patients plasma homocysteine level was significantly lower and serum folate level was significantly higher than the controls However, a statistically significant difference was not detected between groups for mean serum vitamin B12 contrary to other psychiatric disorders 38 Sample Test Result 39 13

14 MTRR Methionine Synthase Reductase 40 MTRR Methionine Tetrahydrofolate Reductase Reductase (Methionine Synthase Reductase) 66 A>G 50% Frequency in Europeans MTRR catalyses methylcobalamin (as a cofactor of MTR) The G allele = risk for: Premature CVD (Brown, et al. 2000) (GG have 1.4x relative risk of CVD compared with AA) Also found to increase risk of NTDs when Vitamin B12 status is low (Botto and Yang 2000) May slow the regeneration of B12 (leading to depletion) 41 Sample Test Result 42 14

15 COMT Catechol O Methyl Transferase 43 COMT Catechol O Methyl Transferase 472 G>A (aka Val158Met) The A allele = 3 4 fold Decrease in enzyme activity! Decreased detox of Catecholamines and Estrogens (Increase NS excitation and estrogen dep. Cancer risk?) High dose 5MTHF with this SNP can = excess Dopamine/Noradr. Supportive Cofactors: Mg, DIM, SAMe, Hydroxy B12 specifically 44 Steroid Hormones Oestrogens & Cancer 45 15

16 Sample Test Result 46 Key Genes Involved In Methylation Gene Meaning Likely Requirements MTHFR MTR MTRR COMT CBS Reduced ability to activate folates for folate methyl cycle 677 Hcysand Cardiovascular System 1298 BH4, NTs and Nervous System Potential B12 depletion (confirm) Increasing B12 requirements Slower regeneration of B12 (Greater need) Higher Catecholamine (Dopamine/Norepinephrine ) levels Altered Sulphur, Taurine and/or Glutathione metabolism Folinic Acid (5 FHTF) and/or 5 MTHF HydroxyB12 w/ Folate or MethylB12 HydroxyB12 preferred esp. if any COMT SNP Hydroxy B12 specifically + Mg, DIM, SAMe P5P form B6 IF required + detox/antiox support 47 Detoxification SNPs CYP1A1 GST M1 GST P1 GST T

17 Revision 49 Detoxification: Phase

18 CYP1A1 Human cytochrome P450 1A1 is one of the most important enzymes participating in human carcinogenesis because it metabolises several pro carcinogens to active carcinogenic metabolites. This meta analysis suggests that polymorphisms of CYP1A1 correlate with increased lung cancer susceptibility and there is an interaction between two genotypes and smoking. In conclusion, this meta analysis points to the A2455G G allele as a risk factor for breast cancer among Caucasian subjects 52 Steroid Hormones Oestrogens & Cancer 53 Sample Test Result 54 18

19 Detoxification: Phase 2 55 GST M1 Glutathione S Transferase M1 Most biologically active member of the GST super family In Liver, responsible for Phase II detoxification of xenobiotics, carcinogens, and products of oxidative stress Gene deletion has a 50% frequency Reduced capacity for hepatic detoxification Increased risk of various cancers, chemical sensitivity, CVD in smokers, atopic asthma, and deficits in lung function Requires a diet rich in antioxidants, minimised exposure to toxins, increase cruciferous and allium vegetables 56 Vegetable Intake & Cancer GST enzyme activity is substantially induced by the metabolic by products of: Cruciferous vegetables (Broccoli, Cabbage, etc...) Supplemental Indoles (I 3 C and DIM) And Allium vegetables (Garlic and Onions) 57 19

20 58 59 Sample Test Result 60 20

21 Steroid Hormones Oestrogens & Cancer 61 GST P1 Glutathione S Transferase P1 313 A>G (aka Ile105Val) Most abundant GST subtype in the lungs and is known to metabolise many carcinogenic compounds Conjugation activity: Wildtype AA = 100% AG = 82% GG = 70% (Watson and Stewart 1998) Polymorphisms associated with increased risk of prostate cancer AG = 1.3x Risk GG = 1.9x Risk (Kote Jarai and Easton 2001) 62 Sample Test Result 63 21

22 GST T1 Glutathione S Transferase T1 Catalyses the conjugation of reduced glutathione to a variety of electrophilic and hydrophobic compounds Gene Deletion in 20% of Caucasians Deletion associated with: An increased risk of lung, larynx and bladder cancers (Hirvonen 1999) As well as brain diseases and skin basal carcinomas in men (Landi 2000) 64 Sample Test Result 65 Antioxidant Capacity (Not Status) SNPs enos SOD2 SOD

23 Antioxidant Capacity Reactive Oxygen Species (aka Free Radicals) are a normal by product of the body s biochemical energy producing processes Highly reactive molecules that can damage DNA, Proteins and Cellular Membranes Antioxidants are synthesised in the body in the form of enzymes to catalyse the neutralisation of these molecules But can also be consumed in the form of various chemical compounds found in a wide variety of foods 67 DNA, Methylation & Oxidation Remember: Any damage to DNA (e.g. via oxidation, radiation etc ) Methylation will be required to repair it Methylation = the way we maintain our genetic material (regardless of what actual genes we have) Many Diseases (esp. Cancer) begin with DNA adducts i.e. risk of maladapted cells or imbalanced proliferation Measuring DNA adducts is arguably the best single assessment of overall cancer development risk in an individual (however only available to specialist research settings thousands of dollars to perform single analysis) The health of our chromosomes is more deterministic of our overall health over time, than any organ 68 enos Endothelial Nitric Oxide Synthase 894 G>T (aka Glu298Asp) Nitric oxide plays key role in the regulation of vascular tone, peripheral resistance and also has vasoprotective effect SNPs effect cleavage of the enzyme, reducing nitric oxide bioavailability in the blood vessel wall, promoting atheroscelerosis T allele frequency = 31% Associated with increased risk of atherosclerosis, essential hypertension, end stage renal disease and pre eclampsia T allele associated with better response to n 3 fatty acids 69 23

24 Sample Test Result 70 SOD2 Manganese Superoxide Dismutase (MnSOD) 28 C>T SOD2 enzyme neutralises ROS produced within cells SNP impacts on distribution of the enzyme within the cell Those without the variant, and with lower consumption of fruits and vegetables, are at increased risk of developing disease, including certain cancers Premenopausal women with the wildtype (CC) have showed an increased risk of breast cancer CC variant = 4.3x Risk 6.0x risk amongst those with the lowest intake of dietary fruits and vegetables (Ambrosone 1999) 71 Sample Test Result 72 24

25 SOD3 Superoxide Dismutase 760 C>G (aka Arg231Gly) Extra cellular form of SOD (major antioxidant enzyme system of the vessel wall) SNP accelerates release of the enzyme from the tissue into the plasma (resulting in significantly reduced tissue SOD3 activity) SNP associated with increased risk for CVD (SOD3 levels are lower in CVD patients) Effect of SNP greatly exacerbated by smoking 73 Inflammation SNPs TNFA IL 6 74 Chronic Inflammation Low grade inflammatory response Prolonged duration is provoked by persistence of the causative stimulus in the tissue Causes tissue damage and is accompanied by simultaneous attempts at healing and repair Extent and time course is variable Depends on the balance between the causative agent and attempts by the body to remove it SNPs have been identified that result in higher basal levels Nutrients & Lifestyle can directly alter genes and resulting Cytokine levels OR address secondary processes 75 25

26 TNF α Tumour Necrosis Factor α 308 G>A SNP has approximately 20% frequency The A allele results in 2 fold increase in TNF α transcription and subsequent TNF α production Associated with low grade chronic inflammation, obesity, obesity related insulin resistance, osteoporosis and altered serum lipid concentrations Omega 3 fatty acids have a particularly anti inflammatory impact in A allele and in those with greater inflammation 76 When interactions between dietary fatty acids and TNF α SNPs on obesity and serum lipid were analysed, both the quantity and quality of dietary fatty acids modulated the relationship between TNF α SNPs on obesity and serum lipid profiles. (Joffe et al. 2013) 77 (Joffe et al. 2010) 78 26

27 (Joffe et al. 2010) 79 Sample Test Result 80 IL 6 Interleukin G>C Key mediator of inflammation, affects adipose tissue metabolism, LPL activity, and hepatic triglyceride secretion CC SNP Frequency = 10 20% in Europeans and 1 2% in Africans C allele carriers have increased circulating levels of IL 6 Associated with elevated CRP levels and CVD Associated with elevated TGs in response to CHO intake and decreased levels of HDL Cholesterol Caucasian women with any C allele showed: decreasing BMI as their Omega 3 intake increased Conversely, as n6:n3 ratio increased, so did BMI (Humphries et al. 2001, Jones et al (2001) 81 27

28 Sample Test Result 82 And Other Gene Results 83 Conclusion Hooray for DNA! * We should all get our genes tested, and prescribe our diets and supplements accordingly 84 28

29 The Big BUT 85 Gene Information Limitations Inability to verify correlations with causations Extremely complex synergy (hard to extrapolate what gene does what in isolation) DNA coding not fully understood (science in its infancy) Indication Interpretation Variable: e.g. A combination of genes may represent a 13% risk of breast cancer but the population average is 14% (i.e. this result SHOULD indicate a BELOW average risk) Testing does not illustrate which SNPs are dominant/expressed 86 Genes Do Not Occur In Vacuum 87 29

30 Resist Genetic Determinism Genes Do Not Dictate Disease! e.g. up to 60% of western population has the gene for diabetes Yet the prevalence of type 1 and type 2 diabetes has increased by 23% and 21% respectively over the past decade! Similar situation with Breast/Prostate Cancer genes etc... Influence of a single SNP may vary widely: e.g. an MTHFR SNP may influence enzyme function 30 60% Yet the SNP with the greatest known effect on human height only accounts for 0.4 % of height variations In the vast majority of cases: Merely having a gene, will not dictate the occurrence of disease 88 Genomics vs Metabolomics Chronic Disease = result of vast multi factorial interactions Full impact of various gene nutrient interactions not yet understood Genomics offers a dynamic approach to revealing the innate relationship between nutrients, genes and chronic disease RISKS Metabolomics offers dynamic assessment of currentdisease ACTIVITY (often before it is evident externally via symptoms) Key: A Gene Always Requires Concert With The Environment & Circumstances To Have Any Relevance! 89 Nutrients and Genes Nutrigenetics Involves SNPs That affect nutrient requirements Nutrigenomics Involves Nutrients That affect gene expression (epigenetics) 90 30

31 Genomics vs Metabolomics (Where Genes Interact With Metabolism) ENVIRONMENT & DIET GENOTYPE NUTRIENTS METABOLISM GENE EXPRESSION CELL GROWTH & FUNCTION PHENOTYPE 91 Methylation In The Body 400+ Enzymatic & Cellular Reactions... DNA Synthesis & Repair Cell Replication & Repair (e.g. fetal development) Neurotransmitter Synthesis and Metabolism Energy Production / Metabolism Hormone Regulation Detoxification Epigenetic Gene Expression / Regulation Telomere Integrity 92 The Closed Loop of Methylation & Genetics We have specific genes that govern our methylation function Yet: ALL DNA itself is comprised of the purine bases synthesised via the Folate Cycle ALL gene expression is regulated via methyl units 93 31

32 Conclusion: Unless you have BOTH a genomic and a metabolic test, you have only one half of the picture and perhaps the lesser half 94 Metabolic Functional Testing 95 Beyond Homocysteine Many factors acting on it! (Vitamin Status, Genetics, Age, Medications..) Reductions have not always demonstrated clear clinical outcomes Risk correlation, but not necessarily causal Having 2 MTHFR variants more common than having elevated Homocysteine 96 32

33 Plasma Methylation Profile 97 Plasma Methylation Profiles Aberrant methionine metabolism can occur in anyone/age A plasma methylation profile provides a functional assessment of the current phenotypic expression (the result of SNPs in concert with the environment) Includes plasma levels of: Methionine, Cysteine, SAM, SAH, Homocysteine and Cystathionine Providing the all important methylation index (ratio of SAM to SAH) These results can better determine appropriate nutritional and lifestyle support for normalising metabolism 98 Key Plasma Metabolites 99 33

34 Cardiovascular Disease evidence (from recent studies) that a higher plasma concentration of AdoHcy is a more sensitive indicator of vascular disease than is a higher plasma concentration of homocysteine 100 Issues With Whole Blood Histamine Seeing as Histamine requires methylation for deactivation, some authors suggest measuring whole blood histamine as a determinant of overall methylation status Yet histamine levels will naturally vary due to endogenous and exogenous metabolic factors (diet, microflora, genes, nutrients, allergens..) normal results vary from lab to lab and country to country, For the purpose of research, a narrow range of whole blood histamine has been classified to represent normal methylation [ mm/l] However, more reliable biochemical indicators of methylation are available: Methylation status can be better measured by either serum Methionine or the ratio of S Adenosyl Methionine [SAMe] to S Adenosyl Homocysteine[SAH]. 101 Plasma Methylation Profile Results

35 Plasma Methylation Profile Results 102

36 Urine Metabolic Tests 103 Metabolic Biochemistry B12 5MTHF P 5 P 400+ Reactions! Glutathione Sulphate Taurine Neurotransmitters

37 Urinary Metabolic (Organic) Acids B Vitamin Status Markers Vitamin B1, B2, B3, B5 Vitamin B12 Vitamin B6 * VMA HVA 5HIAA NT Methylation 106 Key Urine Amino Acids 107 Urinary Amino Acids B Vitamin Status Markers * * (B6) (B6) (B6) (B6) (Folate, B12) * (B6) (B6) (B6) (Folate, B12) (Folate, B12)

38 Urinary Metabolic (Organic) Acids B Vitamin Status Markers Vitamin B1, B2, B3, B5 Vitamin B12 Vitamin B6 * VMA HVA 5HIAA NT Methylation 106

39 * * * Urinary Amino Acids B Vitamin Status Markers (B6) (B6) (B6) (B6) (Folate, B12) (B6) (B6) (B6) (Folate, B12) (Folate, B12) 108

40 Urinary Amino Acids Magnesium Dependant Markers * * 109 Urinary Amino Acids Essential & Non Essential * *

41 Urinary Amino Acids Urea / Ammonia Metabolism * 112 Steroid Hormones Oestrogens & Cancer 113 Steroid Hormones Dried Urine Comp. Hormone Testing

42 Steroid Hormones Dried Urine Comp. Hormone Testing 114

43 CONCLUSIONS 115 Methylation Assessments Useful In Preventing/Supporting: Autism Birth Defects Cancer Cardiovascular Disease Congenital Heart Disease Detoxification Impairment Down Syndrome General Health and Longevity Genetic Disorders Immune Dysfunction Neurodegenerative Diseases Nutritional Deficiencies Psychiatric Disorders 116 Clinical Role of Gene Testing Optimising nutrition Chronic disease prevention / risk management Explain anomalies in specific metabolic processes Targeting diet, lifestyle and supplementation Current interpretation is limited Does not assess epigenetic expression (phenotype) Best used to elucidate only potential genetic influences Cannot incorporate all other factors influencing Methyl function (which are required for appropriate dosing & monitoring)

44 Gene Test Result Summary Must Be Positive Not Deterministic Must use validated SNPs (Well researched criteria for relevance) Must yield interventions that can modify risks (through diet, nutrient and lifestyle changes) Clearly Presented to maximise utilisation and compliance (and patient outcomes) Should be combined with metabolic testing to confirm current status 118 Clinical Role of Metabolic Testing * Determine Current Methylation Needs (Required for Dosing) Urine Metabolite Panels (Organic Acids, AAs): Can indicate the function of the systems affected / needs (e.g. Neuro, GIT, Hepatic, Adrenal/Hormonal ) (e.g. B12 status via Methylmalonic Acid ) Urine Hormone Panels: Estrogen methylation ratio (2OHE1:2OMeE1) Serum Tests: Methylmalonic Acid (B12 Status) Plasma Metabolites e.g. Plasma Methylaton Panel (SAM:SAH) Other std. bloods (with caveats) e.g. Homocysteine, Histamine 119 Test Pairing (Combining Genomics & Metabolomics) (For Methylation Insights) Gene SNPs MTHFR MTR MTRR CBS COMT Metabolic Test Plasma Methylation Panel SAM:SAH Ratio UMP Methylmalonic Acid (B12 Marker) UAA Multiple Methyl Group Markers HDP Liver Phase I& II Markers UAA Multiple Methyl & Sulphur Markers UMP VMA, HVA, HIAA (NT Metabolites) UHP / DUTCH Hydroxyvs Methyl Estrogens

45 How To Dose? For Folate Support: Use Folinic Acid or 5 MTHF For Vitamin B6 Support: Use Pyridoxal 5 Phosphate For Vitamin B12 Support: Use Hydroxy or Methyl Cobalamin * Always combine with rest of B vitamins for proper metabolism But what doses are most appropriate to use in each case? * Note: OVER Methylation can and DOES occur! * TESTING REQUIRED TO ELUCIDATE * (Do not administer doses purely on the basis of assumptions made from genetic test results alone) 121 Include a B Complex B-Complex + Trace Minerals If Nec. Trace Minerals B3 B6 B3 Mg Mg Zn Betaine, Choline B2 B6 B6 122 Methylation Restoration/Optimisation Keys Restore Cell Membrane (Omega 3 EFAs, Choline) Supply Core Nutrients (B Vits esp. B6, B12 and Folate) Supply Sulphur Supportive Nutrients (Taurine, Sulphate, NAC) Assess Current Status/Needs (via Metabolic Testing) Assess Genetic Influences (If needed to elaborate previous) Support Melatonin Release (To Oppose Cortisol and Reprieve Methyl Supply) Reduce Toxic Load (including from choice of supplements)

46 Clinical Rx Tips Getting protocols or RCTs on personalised precision medicine is not going to happen via massive multicentre trials. We need to rethink how we approach EBM If we wait for all studies to be completed first, we are not going to proceed efficiently or even appropriately Using THESE tools is likely to guide the research (rather than the other way round) 124 Thank You for QnA for