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Hereditary fructose i ntolerance and the aldolase B gene

Hereditary fructose i ntolerance and the aldolase B gene. Maddy Ford May 8 th , 2014. What is HFI?. I nability to process fructose. Personal perspective on HFI. “It’s such a part of my life, I don’t think about it much anymore” –Becky Heiser , diagnosed with HFI. What causes HFI?.

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Hereditary fructose i ntolerance and the aldolase B gene

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  1. Hereditary fructose intolerance and the aldolase B gene Maddy Ford May 8th, 2014

  2. What is HFI? Inability to process fructose

  3. Personal perspective on HFI “It’s such a part of my life, I don’t think about it much anymore” –Becky Heiser, diagnosed with HFI

  4. What causes HFI? Defective aldolase B enzyme Glycolytic domain Low complexity region (LCR) Δ4E4 R59OP A149P A174D N334K A337V L256P Molecular Function Biological Process Cellular Component

  5. How does aldolase B protein differ between homologs? Human 364 Glycolytic domain 100% Chimpanzee Glycolytic domain 364 LCR LCR 99% Glycolytic domain Mouse 301 96% 76% 301 Zebrafish Glycolytic domain 65% Fruit fly 361 Glycolytic domain LCR LCR 58% Nematode 366 LCR Glycolytic domain

  6. The protein phylogeny of aldolase B Human Chimpanzee Nematode Fruit fly Zebrafish Mouse

  7. Knowledge gap: Are the C-terminus or LCR important for human aldolase B function? Human 364 100% Chimpanzee 364 LCR LCR 99% Mouse 301 96% 76% 301 Zebrafish 65% Fruit fly 361 LCR LCR 58% Nematode 366 LCR Hypothesis: The C-terminus is important because it contains common mutation sites, the LCR is important for enzymatic folding.

  8. Specific Aim 1: To determine the importance of the C-terminus in fructose metabolism Hypothesis: knockout flies would be unable to process fructose, eventually die Why: Understanding aldolase B structure and function How: RNAi LCR LCR

  9. Aim 1 expected results -/- WT LCR LCR LCR LCR

  10. Is the LCR important for fructose metabolism? Human 100%, 364 a.a. LCR Chimpanzee 99%, 364 a.a. LCR Mouse 96%, 301 a.a. 76%, 301 a.a. Zebrafish 65%, 361 a.a. Fruit fly LCR LCR 58%, 366 a.a. Nematode LCR LCR: ASALAAWGGKAANKKA

  11. Specific Aim 2: To determine the LCR’s importance in fructose metabolism Why: Understanding aldolase B structure and function How: RNAi LCR LCR LCR LCR LCR LCR LCR LCR Hypothesis: all LCR knockouts will be unable to process fructose

  12. Aim 2 expected results LCR LCR LCR LCR LCR could be important for enzymatic folding LCR LCR LCR LCR

  13. Can we find a substitute for aldolase B? Aldolase B is unstable  Find a substitute to mimic its action

  14. Specific Aim 3: To identify chemicals that mimic aldolase B, and what genes they affect. Why: Investigating HFI drug possibilities How: Chemical genetics Hypothesis: Drugs similar to aldolase B in structure and function will rescue WT phenotype, increase glycolytic gene expression

  15. Aim 3 expected results Glycolytic gene expression Effective drug Ineffective drug Glycolytic gene expression

  16. Conclusions Further testing of the aldolase B gene and protein could provide new insight on enzyme activity Chemical genetics could yield a drug to allow individuals with HFI to consume fructose without experiencing symptoms LCR LCR

  17. Future directions Determine the importance of Alanine within the LCR: ASALAAWGGKAANKKA Researching similarity between mouse and zebrafish Further chemical testing

  18. References 1. Cox, T.M. (1994). Aldolase B and fructose intolerance. The FASEB Journal, 8(1), 62-71. 2. James, C.L., Rellos, P., Ali, M., Heeley, A.F. & Cox, T.M. (1996). Neonatal screening for hereditary fructose intolerance: frequency of the most common mutant aldolase B allele (A149P) in the British population. Journal of Medical Genetics, 33(10), 837–841. 3. Kajihara, S., Mukait, T.,  Arai, Y., Owada, M., Kitagawa, T. & Hori, K. (1990). Hereditary Fructose Intolerance Caused by a Nonsense Mutation of the Aldolase 4. Understand Dairy Sensitivity: Overview. Lactaid. Accessed 13 April 2013. <http://www.lactaid.com/dairy-sensitivity> 5. Ekker, S.C., and Larson, J.D. (2001). Morphant technology in model development systems. Genesis 30:89–93. 6. Miller, D, Hannon, C, and Ganetzky. (2013). A mutation in Drosophilaaldolase causes temperature-sensitive paralysis, shortened lifespan, and neurodegeneration. Journal of Neurogenetics. 26 (3-4): 317-327. doi: 10.3109/01677063.2012.706346. 7. Thurston, J.H., Jones, E.M., Hauhart, R.E. (1974). Decrease and Inhibition of Liver Glycogen Phosphorylase After Fructose. Diabetes, 23 (7), 597-604. doi: 10.2337/diab.23.7.597 8. NCBI BLAST. Homo sapiens chromosome 9, GRCh37.p13 Primary Assembly compared to Musmusculusaldolase B, fructose-bisphosphate, mRNA (cDNA clone MGC:36362 IMAGE:4975382), complete cds. http://blast.ncbi.nlm.nih.gov/Blast.cgi

  19. Image references • Baby with fruit. http://en.wikinoticia.com/lifestyle/Maternity/108058-feeding-children--juice • Human liver. https://www.sccollege.edu/StudentServices/HealthWellnessCenter/AlcoholEffects/Pages/Liver.aspx • Fructose. https://ketopentose.wordpress.com/page/3/ • Glucose. http://science.halleyhosting.com/sci/soph/energy/resp/notes/krebs.htm • Cytosol. http://education-portal.com/academy/lesson/cytosol-definition-function-structure.html#lesson • Aldolase B in glycolysis. http://david-bender.co.uk • Clustal Omega logo. http://www.clustal.org/ • Clustal Omega sequence aligntment. http://www.ebi.ac.uk/Tools/msa/clustalo/ • Drosophila. http://obbard.bio.ed.ac.uk/photo_gallery/Drosophila_kuntzei_lab.html • Drosophila on fruit. http://en.wikipedia.org/wiki/Yeast_in_winemaking • Strawberry. http://www.figtreeolivebranch.com/how-to-hull-a-strawberry/ • C. elegans. http://fangyenlab.seas.upenn.edu • Fruit fly on fruit. http://www.usaprepares.com/health/strawberry-alert-the-2mm-long-asian-fruit-fly-that-could-decimate-britains-fruit-industry • Lactaid. http://www.drugstore.com/lactaid/qxb7681 • Morpholinos. https://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMorpholino.shtml • Fly screen. http://hms.harvard.edu/news/fly-screen-3-13-14 • Business cat. http://beta.diylol.com/memes/1-business-cat/posts/232916-business-can-wait-now-it-s-time-for-your-questions

  20. Questions?Thanks for listening.

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