cystic fibrosis

Question 1

indentifiers

Answer 1

very salty tasting skin
appetite but poor growth and weight gain (failure to thrive)
coughing, wheezing + shortness of breath
abnormal bowel movements
shows recessive pattern in familial history

Question 2

genetic linkage

Answer 2

linkage involves 2 or more genes or loci which are located on the same chromosome in a linear fashion
linkage usually involves genes or loci which are located close to each other
goal is to place genes and genetic markers along chromosomes, order them and design genetic map
genetic markers- sequence of dna with unknown functions but easily recognised as landmarks
smaller amount of recombination observed between genes = more tightly linked genes

Question 3

CF linkage analysis

Answer 3

looking for polymorphic markers
serum arylesterase paraoxonase (PON)
looked at normal danish familes and danish and english cf families
highest correlation found between inheritence of paroxinase and CF
linkage studies for PON against 64 other polymorphic marker systems did not give such a close relationships

C0CRI-917- colocalised with PON and CF gene
location of CF gene narrowed to about 1% of human genome
testing for other markers that overlapped PON and D0CRI-917 was performed- provided 2 closer markers Met and D7S8
further analysis provided markers D7S340 and D7S122
enabled chromosome jumping + mapping

Question 4

CFTR protein

Answer 4

locus- 7q31.2- CTFR gene found in region q on long arm of human chromosome 7
gene is about 200,000 bp long, contains 27 exons
mRNA- intron free mRNA trancript for CTFR gene is 6129bp long
protein size- CTFR protein is 1480 aa long

Question 5

CTFR candidate region

Answer 5

cystic fibrosis transmembrane regulator
polymorphic dna (D0CRI-17) was linked to PON locus
marker linked to CF within families using southern blotting and linkage analysis
RFLP (restriction fragment length polymorphisms) markers located on chromsome 7 were tested for linkage to CF + loosely linked genetic marker was found
put into vectors + grow bacteria to analyse
by in situ hybridisation- marker mapped to chromosome 7

Question 6

gene mapping

Answer 6

Chromosome walking/jumping used to identify
- 7 jumps of 50-75 kb were made with each arrival point then serving as a new origin for chromosome walking
- Narrow the region of exploration and cloning of the CF gene

Question 7

CFTR defects

Answer 7

cystic fibrosis caused by defets in CFTR protein
CFTR helps to maintain balance of salt and water on surfaces of the body
CTFR is responsible for reabsorption of chloride (and sodium) in reabsorption duct of sweat gland but dysfunction of CTFR leads to hypertonic beads of sweat seen in CF

Question 8

CF inheritence

Answer 8

autosomal recessive
early age of onset- average life expectency 30 yrs
approx 1 in 2000 new births affected
if both parents carry mutation- 25% child will get CF

Question 9

CFTR

Answer 9

3 base pair deletion at codon 508
deletes a Phe residue but maintains reading frame
most common is deletion of phenylalanine (f508) in 70% of CF cases
over 2500 mutations of CF found
F508 accounts for 70% of cases

Question 10

F508 mutation benefits

Answer 10

mutation occured over 500,000 ya in northern europe
seen more commonly in caucasians
individuals with 2 copies of F508 get cystic fibrosus and often cant reproduce
having 1 copy of F508 reduced water loss during cholera, increasing survival
CF heterozygotes possess selective advantage of resistance to cholera

Question 11

genetic screening

Answer 11

patients with raised IRT (Immunoreactive trypsinogen) are screened for 4 most common pathogenic variants in northern eurpoean populations
p.Phe508del, p.Gly542X, p. Gly551Asp and c.489+1G>T,
use real time genotyping pcr accounting for 80.6% of pathogenic variants in northerm eurpean caucasian populations
if one pathogenic variant is found, further screening is undertaken

Question 12

CF pathophysiology

Answer 12

altered chloride channel alters mucus production, resulting in a more viscous soltion
effects all mucosal membranes
results in mucus retention and chronic infection, local airway inflammation that is harmful to the lungs
measure of lung function is FEV1 - key predictor of life expectancy
CF affects several body systems + comorbidities can occur in pancreas, liver, sweat glands + vas deferens
Cf is a multi system genetic disorder

Question 13

CF symptoms

Answer 13

malabsorption and failure to thrive from birth
reccurent/ persistent chest infections
pancreatic insufficiency
meconium ileus- substance in small intestine that is normally broken down, but is not in CF due to pancreatic insuffiency so may cause obstruction

Question 14

role of CFTR

Answer 14

fluid homeostasis is tightly regulated in epithelial tissues wehre CFTR anion channel plays a major role in regulating both secretion and absoprtion covering a broad range of epithelial tissues including sweat + salivary glands etc
CFTR regulates many mechanisms in epithelial physiology such as maintaining epithelial surface hydrated and regulating luminal pH
trnascellular salt secretion is mainly controlled CL- exit across the apical memabrane and this occurs predominatly via CTFR

Question 15

sweat test

Answer 15

measures the conc of chloride and sodium that is excreted in sweat
2 relaible positive results on 2 separate days is diagnostic for CF
clinical presentation, family history and patient age must be considered to interpret the results
non cf= <60mmol/l CF=>60mmol/l

Question 16

diagnostic criteria

Answer 16

at least one of
1. one or more clinical manifestation- salt loss syndrome, chronic bronchitis, pancreatic insufficiency
2. positive newborn screening test
3. history of CF in sibling
4. elevated sweat chloride test
5. idenitification of a mutation in each CTFR gene known to cause CF

Question 17

CF lung disease symptoms + treatments

Answer 17

increased cough with sputum production
hemoptysis
increased shortness of breath
reduction in FEV1
worsening infiltrates on chest x ray

treatments
antibiotics to fight lung infections
bronchodilators
enzyme supplements
tobramycin

Question 18

mucolytic therapy

Answer 18

DNase (pulmozyme)- chronic use improves FEV1 and causes fewer exacerbations - used to thin mucus
mucoactive therapy reduces viscoelasticity of sputum from patients with CF and enhances clearance of secretions
only benefits some patients

Question 19

bronchodilators

Answer 19

chronic use improves FEV1 in some patients
B andrenergic agonists, anticholinergic agnets

Question 20

CFTR genetic analysis

Answer 20

mutation screening panels
US= preconceptual and prenatal carrier screening for CF
UK= national screening commitee
1. patients with raised IRT are then screened for 4 most common CFTR pathogenic vairnats (northern european cauc)
2. NHS genomics screen for p.Phe508del, p.Gly542X, p. Gly551Asp and c.489+1G>T
3. if one pathogenic variant is found further screening undertaken using ARMS methodology

current commerical screening tests look for presence of between 25-30 mutations- detect CF allele only 90% of time
clinical phenotypes can vary widely across mutations - one allele makes large difference

Question 21

phenotypic penetrance

Answer 21

can look at relationship between phenotype, genotype and CFTR function in patients with CF
number of channels operating tells efficiency of channels
sweat chloride and CFTR funtion linked
100mmol/l CL = 0% CFTR function
30mmol/l cl= 50% CFTR function
<30mmol/l cl = 100% function

Question 22

classes of mutation

Answer 22

I- CFTR is not synthesised
II- CFTR is inadequatley processed (misfolding)
III- not regulated (no agonist response)
IV- shows abnormal conductance
V- has partially defective production
VI- displays accelerated degradation

mutation I,II V and VI result in absence of reduced quantity of CFTR protein at the cell membrane wehreas III and IV influence the function or activity of CFTR at the cell membrane
mutation 1- greatest disruption of CFTR mediated cl transport
chloride transport increases through the remaining classes
function below 3%= symptoms of CF

Question 23

F508 deletion

Answer 23

results in multiple protein defects
defect in CFTR processing and trafficking- few to no CFTR channels are present at the apical cell surface
F508 del is class 3 mutation with reduced channel opening probability
manifests characteristics of class 6 mutation so the few CFTR proteins that reach cell surafce have decreased surface stability
most common CFTR mutation in world

Question 24

G542X

Answer 24

mutations results in defective biosynthesis of CFTR protein
nonsense mutation which produces a premature stop codon
cell cannot synthesise a full length CFTR protein
class 1 mutation
2nd most common mutation
results in little or no CFTR activity

Question 25

personalised medicine

Answer 25

CFTR modulators- potentiators, correctors and premature stop codon suppressors
personalised therapies rely on extensive knowlege of CFTR mutation clasaes and focuses on targetting certain mutations with specifc molecules
only individuals with specific mutations can be treated with traget drugs

Question 26

future therapies for CF

Answer 26

gene therapy to introduce the wild type CFTR via non viral liposomal vector- delivering copies of coding sequence of normal CFTR dna to target cells
genome editing- facilities knock out or knock in mutations by introducing a double strand break in genome