Essay marking scheme0: No answer2 : 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 Core5: 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.

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

Genetics of cystic fibrosis

Cystic fibrosis (CF) commonest monogenic disease1 in 2500 Caucasian births

1 in 15 carriers7000 people in UK

Autosomal recessive diseaseMost common (~70%) phe506del (F508)>1600 other mutations

The sweat gland and CF

Quinton 2007 Physiology

“a child that taste salty when kissed will soon die”Alonso y de los Ruyzes 1606

The sweat gland and CF

Quinton 2007 Physiology

CF is primarily a lung disease…

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• 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 (15th Dec, 2007) BMJ for more extensive symptom list

Lung disease in CF

Life expectancy

<40 yrs

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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

The CF Transmembrane Conductance Regulator protein

CFTR 1480 amino acidsMember of ABC family

The CFTR protein

CFTR – how does it work?

Not a classical ABC transportercAMP regulated Cl channelRole of ATP?

Mutations in CF

Mutations in CFTR can result in trafficking and/or function defects Class IV

Defectiveconduction

Class IIIDefectiveregulation

Class IIDefectiveprocessing

Class IDefective protein production

Class VReduced mRNA and/or protein often due to splicing defects

F508 is the most common CF mutation

F508 is primarily a class II mutationAccounts for ~70% of CFProtein 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’

Nature, 1992

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

‘Correctors’ increase surface expression

Chemical chaperones: e.g. glycerol, taurine, can suppress protein-folding defectsDisadvantages: 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

‘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…

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…

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

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?