1. Essay marking scheme 0: No answer 2 : Fail. Very incomplete or very inaccurate answer, or answer indicating substantial and potentially dangerous misunderstanding. 3: Borderline Fail. Poor attempt, inadequate in some respects (for example, with inaccuracies, omissions or irrelevant information) 4: Borderline Pass: information is relevant and largely accurate, but indicating little more than a reasonable understanding of the Core 5: Pass: answers with good (and relevant) content extending significantly beyond the Core Answers scoring 5 can be given up to three bonus marks : 1 mark: for a well-organised, clear and direct answer to the question, 1 or 2 marks: for the following as they may be possible according to the question set: the appropriate use of experimental evidence, reference to relevant clinical matters, an integrative (as opposed to a narrow) approach, a perceptive/original and relevant discussion.

2. The CF Transmembrane Conductance Regulator protein and cystic fibrosis Syllabus: 1.6.5 Transporter structure and function 8.2.4 Tissues of respiratory system 9.5.3 GI secretion 9.5.5 Intestinal electrolyte transport 14.7.4 Secretin control via cAMP of Cl-/HCO3- exchange

3. Genetics of cystic fibrosis Cystic fibrosis (CF) commonest monogenic disease 1 in 2500 Caucasian births 1 in 15 carriers 7000 people in UK Autosomal recessive disease Most common (~70%) phe506del (  F508) >1600 other mutations

4. The sweat gland and CF Quinton 2007 Physiology “a child that taste salty when kissed will soon die” Alonso y de los Ruyzes 1606

5. The sweat gland and CF Quinton 2007 Physiology

6. CF is primarily a lung disease… Livraghi & Randell 2010 Toxocol Path

7. …but not exclusively Pancreas –Exocrine –Endocrine (CF related diabetes) GI tract –Constipation due to thickened faeces –Malabsorption of vitamin D  osteoporosis Liver (cirrhosis) –Thickened bile Infertility in males –vas deferens absent See Davies et al (15 th Dec, 2007) BMJ for more extensive symptom list

8. Lung disease in CF Life expectancy <40 yrs Davies et al 2007 BMJ

9. Diagnosis of CF All babies are given Guthrie blood spot test measures ↑ immunoreactive trypsinogen if positive  genetic screening & sweat testing Advantages of early diagnosis: nutritional benefits, early access to care, relatively quick diagnosis, appropriate counselling

10. The CF Transmembrane Conductance Regulator protein CFTR 1480 amino acids Member of ABC family

11. The CFTR protein

12. CFTR – how does it work? Not a classical ABC transporter cAMP regulated Cl channel Role of ATP?

13. Mutations in CF Mutations in CFTR can result in trafficking and/or function defects Class IV Defective conduction Class III Defective regulation Class II Defective processing Class I Defective protein production Class V Reduced mRNA and/or protein often due to splicing defects

14.  F508 is the most common CF mutation  F508 is primarily a class II mutation Accounts for ~70% of CF Protein retained in ER and then degraded by proteosome F508 is in NBD1, and its mutation makes the domain less temperature sensitive can increase surface expression by reducing temperature (Denning et al 1992) ‘correctors’

15. Nature, 1992

16. What was the rationale for looking at the effects of temperature? What is the potential clinical significance of the study? What cells were used? What are the key results from figure 1, 2 and 4? Questions

19. ‘Correctors’ increase surface expression Chemical chaperones: e.g. glycerol, taurine, can suppress protein-folding defects Disadvantages: need high (mM) concs as non- specific effect ‘Correctors’ act as chaperones to rescue cell surface expression More specific correctors have been found by screening of small molecule libraries (pharmacochaperones) Work by enhancing folding, decreasing degradation, increasing plasma membrane stability

20. ‘Correctors’ 2. Targeting endogenous molecular chaperones Could try to either -Pharmacologically decrease interaction with chaperones that signal degradation -Knock down chaperones with antisense RNA Mixed/controversial results…

21.  F508 is also a class III mutant Even when at the membrane, the conductance of  F508 is lower than WT Need potentiator (drug which increases conductance) - act directly on CFTR - open basolateral K + channels - open an alternative route for apical Cl - exit Pharmacology currently seen as more feasable route that gene therapy…

22. Difficult to target specific cells, cell turnover relatively high needing continual treatment UK Cystic fibrosis Gene Therapy consortium performing clinical trials

23. Reading primary literature Introduction –What was known and not known –Why did they do the experiment –What is the fundamental question addressed by the experiment Methods Techniques –Overview of techniques; what did they use? –What are their limitations? Results –Each figure is a mini-paper: state why they did it, what was the technique, what the results show, what do the results mean Discussion –Overall merit of paper; clinical significance of findings –What remains to be done?