Genetically Modified Soybeans: Equal Allergenicity as their Wild Type Counterparts?

1. Genetically Modified Soybeans: Equal Allergenicity as their Wild Type Counterparts? Katie Van Den Einde November 24, 2009 Advisor: Dr. Chastain

2. Overview • Introduction: GM foods, allergies, controversy • Paper 1 • Paper 2 • Paper 3 • Paper 4 • Current regulations • Conclusions

3. Importance • GM foods: • Soybeans • Corn • Tomatoes • Rice • Canola • Potatoes • Sugar beets • Sugar cane

4. Modifications • Herbicide resistance • Insect resistance • Disease resistance • Addition of proteins/vitamins • 2003 – 84% of US soybean acreage was glyphosate tolerant (Roundup® ready)

5. Basics of Genetic Modification • Procedures • 1. Plasmid insertion • 2. Gene “guns” • 3. Protoplasts

6. Allergies • Majority of allergic reactions are immunoglobulin E (IgE) mediated. • IgE allergies affect about 1-2% of adults • 2-8% of children

7. Symptoms: • Itchy, watery eyes • Rash • Congestion • Itchiness • Difficulty breathing • Anaphylactic shock (Can be life threatening)

8. Basics of allergic reactions • 1-Allergen • 2-IgE antibodies • 3-Mast cells • 4-Histamine release

9. Anti-Histamines

10. GM Controversy • Ethics • Gene flow • Resistance • Harm to other organisms • Allergens???

11. Overview • Introduction: GM foods, allergies, controversy • Paper 1 • Paper 2 • Paper 3 • Paper 4 • Current regulations • Conclusions

12. Paper 1: • Identification of a Brazil-nut allergen in transgenic soybeans • New England Journal of Medicine 1996

13. Purpose: • To assess ability of proteins from • 1)soybeans (Glycine max) • 2)transgenic soybeans • 3)Brazil nuts (Bertholletiaexcelsa) • 4)purified 2S albumin to bind to IgE serum

14. Methods: • Radio allergosorbent test (RAST) – 4 serums • Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) – 9 serums • Skin Prick Tests

15. RAST basics

16. Results: RAST More inhibition of IgE binding = more allergic. Triangles= WT Squares= GM soybean Circles= Brazil nut

17. Results: SDS-PAGE Total Proteins IgE binding IgE binding 2S 2S Standards WT SB TG SB Brazil Nut 2S in TG SB Brazil Nut WT SB TG SB Brazil Nut

18. Results: Skin-Prick Test

19. Main Points: • GM soybean protein successfully competed with Brazil nut protein. • IgE from 8/9 allergic to Brazil nut bound to introduced 2S albumin in GM soybeans.

20. Overview • Introduction: GM foods, allergies, controversy • Paper 1 • Paper 2 • Paper 3 • Paper 4 • Current regulations • Conclusions

21. Paper 2 • Lack of detectable allergenicity of transgenic maize and soya samples Journal of Allergy and Clinical Immunology 2005

22. Purpose: • Monitor 5 GM products whose transgenes came from sources with no allergenic history

23. Methods: • Food Survey • Previous exposure? • Skin Prick Tests • 27 kids with food allergies • 50 patients with asthma rhinitis • SDS-PAGE

24. Flour products tested

25. Food survey results

26. Western Blot

27. Testing Lab Supply Western Blot SDS PAGE

28. SDS PAGE Western Blot

29. Skin prick and IgE results

30. Main Point: • No detectable difference in IgE reactivity between wild type and GM soybeans or corn.

31. Overview • Introduction: GM foods, allergies, controversy • Paper 1 • Paper 2 • Paper 3 • Paper 4 • Current regulations • Conclusions

32. Paper 3 • A comparative study of the allergenic potency of wild-type and glyphosate-tolerant gene-modified soybean cultivars • Acta pathologica, microbiologica et immunologica Scandinavica 2003

33. Purpose: • To compare allergenicity of 8 wild type and 10 GM soybeans varieties (all for CP4 EPSPS)

34. Methods: • RAST (serum from 10 patients) • SDS-PAGE • Histamine Release test • Skin prick tests

35. RAST results More inhibition of labeled IgE binding = more original serum bound first.

36. RAST results Concentration of extract needed for 50% inhibition of IgE binding (variety #12)

37. Histamine Release results Histamine Release (0=negative, 6=lots) Skin Prick Test Notice lack of any major differences – no where to point an arrow!

38. Histamine Release for patient I Pretty similar!

39. Main Points: • Difference between patients’ response, but no statistical difference between WT and TG soybeans. • Addition of CP4 EPSPS gene ≠ higher allergenicity

40. Overview • Introduction: GM foods, allergies, controversy • Paper 1 • Paper 2 • Paper 3 • Paper 4 • Current regulations • Conclusions

41. Paper 4 • Genetic modification removes an immunodominant allergen from soybean • Plant Physiology 2003

42. Purpose: • To silence the Gly m Bd 30K (P34) gene transgenically

43. P34 • A major soybean allergen • More than 65% of soy-sensitive patients react only to the P34 protein • Less than 1% of total protein • Pigs, calves and salmon also allergic

44. Methods: • Created a P34 silencing vector (plasmid pKS73) • Grew these into homozyous strains • Used SDS-PAGE for presence of P34 protein

45. Results Monoclonal antibodies

46. Soybean Protein “Map”

47. Protein Analysis Missing P34 proteins and intermediates Wild type P34 Silenced

48. Main Points: • TG and WT were indistinguishable in size, shape, protein and oil content • P34 gene silencing was successful

49. Overview • Introduction: GM foods, allergies, controversy • Paper 1 • Paper 2 • Paper 3 • Paper 4 • Current regulations • Conclusions

50. Who’s in charge? • Coordinated Framework for the Regulation of Biotechnology - 1986 • 3 regulatory bodies of genetically modified foods: (Animal and Plant Health Inspection Service)