cystic fibrosis

Question 1

general information

Answer 1

• a monogenic disease
• fibrocystic disease of the pancreas
• a malabsorption syndrome with chronic pulmonary infection
• median survival >40 years.
• carrier frequency 1 in 25 in Caucasian population
• autosomal, recessive disease

Question 2

different CF mutations

Answer 2

3 quarters of pateints hace same mutation: F508 in CFTR [a deletion of phenylalanine 508]. over 200 other CT mutations known.

• screening for F508 is by PCR amplification of relevant CFTR region.
• mutation frequencies vary geographically

Question 3

clinical presentations

Answer 3

respiratory symptoms, often associated with respiratory infection.
malnutrition. steatorrhea. intestinal obstruction. electrolyte imbalance.

Question 4

FVC- forced vital capacity

Answer 4

the amount of air that can be exhaled from a deep breath. in CF mean FVC is ~85% normal

Question 5

FEV1

Answer 5

forced expiratory volume in 1 sec [of FVC test]. in CF mean FEV1 is ~72% of normal

Question 6

CFTR gene

Answer 6

located on long arm of chromosome 7. encodes CFTR- the 1480aa cystic fibrosis transmembrane conductance regulator. chloride transporter

Question 7

what is CFTR

Answer 7

an epithelial chloride channel:

• Na+ reabsorption normally accomplished by an active Na+,K+-ATPase
• failure to transport Cl- in the same direction as Na+ results in membrane hyperpolarization leading to reduced Na+ transport. sweat of sufferers tastes salty

Question 8

possible defects in CFTR

Answer 8

• defective assembly/ folding leading yo reduced cell-surface protein levels. lack of CFTR leads to severe effect
• failure of chloride channel opening- defective ATP binding- dyregulation of channel opening
• altered chloride transport- mutations in membrane-spanning region result in symptoms of variable severity

Question 9

delta-F508 mutation

Answer 9

deletion of 3bp [CTT] in 10th exon of the CFTR gene. product is missing single aa [i.e. no frame-shift] but stability and surface expression significantly reduced.

delta is shorthand for deletion

Question 10

PCR test for delta-F508 mutation

Answer 10

WT- 97bp
heterozygote carrier- 97bp and 95bp
homozygote CF sufferer [94bp].
if negative for this test, chanced of being CF carrier reduced to 1:100

Question 11

amplification refractory mutation system [ARMS]

Answer 11

relies on the absolute requirement for complementary at 3’ end of PCR primers. primers match: product formed and vice versa

Question 12

PCR basic steps

Answer 12

1) denaturation: 2 sreands of DNA separated [melt down] to form single stranded DNS
2) annealing: annealing of primer to each strand
3) extension: DNA polymerase adds dNTPs complementary to templates strands at 3’end of primer.
4) step 1-3 repeat cycle 35-40 times

Question 13

a primer

Answer 13

a primer is a short oligonucleotide which is the reverse complement of a region of a DNA template. it would anneal to a DNA strand to facilitate the amplification of the targeted DNA sequence

Question 14

designing a PCR primer

Answer 14

1) primers are complementary to their target sequences, and both forward and reverse primers should be written 5’ to 3’
2) primers tend to be >15nt
3) the part that matches the target sequence should have a melting temperature [Tm]
of around 50-65C
4) [A+T]2 + [G+C}*4=Tm in degrees centigrade