Week 9

Question 1

CF (Overview)
> MOST COMMON, SEVERE AR disorder
> FIRST GENETIC DISORDER elucidated by POSITIONAL CLONING

Answer 1

1. Linkage: 7q31.2
   > Closely linked flanking markers (oncogene MET and polymorphic locus D7S8)
   > MET…CF…D7S8
2. Mostly due to delta Phe508 mutation

Question 2

CF: Pulmonary/Respiratory

Mucus-blocked airways and bacterial infections (green)

Answer 2

1. Co-morbidity: Lung
   > Trachea –> bronchi –> bronchioles –> alveoli ducts –> alveoli
   > CF: mucus plugging in alveoli and pancreatic duct; airway dilation; lung blocked by inflammation and secretions
   > Initial precaution: chest percussion; ventilator

Question 3

CF: GI + Pancreatic

Blocked ducts, decrease in enzymes

Answer 3

1. Pancreas: endo/exocrine
   > Endo: insulin, glucagon, and Islets of Langerhans
   > Exo: duct cells secrete NaHCO3; acinar cells secrete digestive enzymes
   > CF: duct dilation, eosinophilic/acid dye mucin plugging; exocrine acini degeneration and replacement with fibrous tissues; no digestive enzymes –> fat tissue buildup; abnormal IoL

Question 4

CF: Sweat glands (Salty Baby Syndrome)

Dehydration; heat exhaustion/fever

Answer 4

1. Normal: NaCl and H2O out; NaCl reabsorption
2. CF: defective CFTR Cl- channel
   > No Cl- Reabsorption
   > Isotonic secretion intact, but reabsorptive duct has hypertonic sweat

Question 5

CF: Reproductive

Block/absence of Vas deferens and Aspermia; cervical polyp

Answer 5

1. May occur in males without CF symptoms (one mild and one severe CFTR mutation; if both mild –> no CF observed)
2. Derivatives of mesonephric duct structures sensitive to CFTR dysfunction
3. GWAS–fertility related genes: Polymorphism is a valine or methionine in the same position
   > Valine residue –> male birth rate

Question 6

CFTR Gene

Answer 6

1. Transcript and regulator (250 kb, 27 exons, 1480 AA)
2. ATP-dependent (cAMP) Cl- channel; regulates ENaC (epithelial)
   > Transmembrane domain (TMD); nucleotide binding fold (NBF; ATP-binding site); regulatory domain with Cl- passing through
   > NORMAL: 2 Cl- channels (cAMP and Ca2+ activated) with normal mucin + HCO3- discharge
   > CF: decreased Cl- secretion and compensatory Na+ influx to retain ELECTRONEUTRALITY + water influx –> DEHYDRATION + STICKY MUCUS (mucin has no HCO3-; molecules adhere to each other condensely and viscously; intracellular cation shielding from Ca2+ and H+ but extracellular e- repulsion from HCO3-

Question 7

Class 1 CF

Answer 7

Splice Mutation intron 4 donor site G –> T
> No protein due to premature stop codon, but normal mRNA
> Deal: rescue protein

Question 8

Class 2 CF

Answer 8

Defective processing with folding due to delta F508
> Made but immediately destroyed due to deletion
> Deal: correct folding
>*** Defective protein retains Cl- channel function normally in cell-free lipid membranes, but once detected as MISFOLDED, it is DEGRADED shortly after synthesis via proteasome pathway before reaching crucial site (ER) at cell surface

Question 9

Class 3 CF

Answer 9

Defective Regulation with gate opening

> Deal: restore channel conductance

Question 10

Class 4 CF

Answer 10

Defective conduction due to alteration of Cl- channels
> Correct opening, but no material passage
> Dea: Restoring channel conductance

Question 11

Class 5 CF

Answer 11

Reduce transcript copies
> Not enough proteins produced
> Deal: maturation and correct mis-splicing

Question 12

Class 6 CF

Answer 12

Cell surface instability
> Protein cannot remain in membrane long enough
> Deal: promoting protein stability

Question 13

Gene Targeting for CF (KO and KI)

Answer 13

1. KO: chimeric + normal –> crossbreed (40% homozygous for disrupted delta F508 displays CF abnormalities in adult human diseases)
   > test for death resulting from intestinal obstruction before 40 days of age

**KO CFTR demonstrates that delta F508 is responsible for CF, and KI (F508 Deletion) mimics human disease with this mutation and identifies potential treatment option

Question 14

CF Treatments (No curative so far)

Answer 14

1. Objectives:
   > Pulmonary secretion clearance, control of infection, pancreatic enzyme replacement, adequate nutrition, prevention of intestinal obstruction
2. Respiratory failure –> lung transplantation (only option)

Question 15

CF: Science to Clinical Trials

Answer 15

1. Aminoglycosides (Antibiotics) bind to ribosomes and terminate translation
   > Low [Ab] can cause stop codon skipping (10% CF due to premature stop codon)
   > G418: Arg553Stop; Gentamicin converts it to 553Tyr (nearly normal CFTR); CLASS I
2. Ataluren: allows ribosomes to read stop codons
3. Ivacaftor: CLASS III
   > one copy of Glycine –> Aspartic acid mutation at 551 in CFTR; G551D can be at surface, but once there protein cannot transport Cl- through channels (Ivacaftor improves this by binding to channels to induce non-conventional gating mode)
4. Lumacaftor (corrector):
   > Partially restores CFTR function in homozygous F508del by suppressing protein folding defects via enhancing NBD1 + TMD1/2 interactions (CLASS II)
5. Orkambi (3+4):
   > For homo F508del: I increases CFTR protein activity at surface; Ls helps CFTR folding and Is increases CFTR proteins in PM