CF carrier frequency?
1/25
CF incidence?
1 in 2500 live births
Where is CFTR and what size?
7q31.2, 27 exons
What cells are affected in classical CF?
Mucus-producing cells in exocrine glands
Organs which are affected?
Lungs and respiratory tract, pancreas, gut, male genital tract
Condition which occurs in 10-20% of newborns with known CF?
Meconium ileus- failure to pass meconium due to “blocked” intestine
Diagnostic requirements for CF?
One or more phenotypic feature + either 2 known disease causing mutations, or 2x sweat test with Cl greater than 60
Main cause of morbidity and mortality?
Pulmonary disease
Mean survival?
40 years (56 years if pancreatic function intact)
Genotype phenotype correlation:
a) pancreatic sufficiency
b) pulmonary disease
b) male infertility
a) good
b) poor
c) good
Strcture and function of CFTR protein?
Three domain intramembrane protein which regulates movement of Cl ions
Commonest mutation in:
a) white UK
b) Ashkenazi jews
a) F508
b) W1282X (nonsense)
Majority of CF mutations are what type?
Single nucleotide changes
Classes of CF mutation? (1-5)
1- affects translation (G542X)
2- affects protein maturation (F508del)
3- affects function of protein at cell surface
4- causes a decrease in Cl conductance (R1347P)
5- cause decrease in synthesis of CFTR
Mutation classes associated with a) classical phenotype b) milder phenotype
a) 1, 2, some 3
b) 4, 5, some 3
Why are CF mutations common? (2)
Founder effect- loss of genetiuc variation that occurs when a new population is established by a small number of individuals from a larger population Heterozygote advantage- when the heterozygote has a greater relative fitness than either class of homozygote
CF-related disease which may be seen in men with CF, and in those who have mutations in CFTR gene but don’t present as classical CF
Congenital bilateral absence of the vas deferens (CBAVD)
Partially-functioning allelels associated with CBAVD?
R117H or 5T
What should be checked when “obstructive azoospermia” is written on the reqest form, or when R117H has been identified?
poly T status
What affects the efficiency of splicing adjacent to the exon 9 acceptor splice site?
Tract of thymidine residues which can be 5, 7 or 9 long. 5T is least efficient- reduced production of functional CFTR protein. 9T is the common allele
Combinations of thymidine residues and mutations:
a) F508del on one chromosome, R117H in cis with 5T
b) F508del on one chromosome, R117H in cis with 7T
c) 5T on one chromosome, 5T or CF mutation in trans
d) F508 on one chromosome, R117H in cis wit 9T
a) classical CF
b) no symptoms or should show CF-related disease
c) benign or could show CF related disease
d) rarely found, benign
F508del is usually found in cis with…
9T
What other site affects splicing efficiency? What is the commonest size of this?
Upstream of exon 8 polyT, a TG repeat tract. 11 repeats is normal
What is the worst combination involving TG repeats?
12 or 13 repeats in cis with FT on one chromosome, severe CF mutation in trans
Testing for CF?
ARMS
Which CF kit can test poly T status?
CFEU2 (the other one is CF29)
Mutations tested by CF29?
29 mutations, uses gel electrophoresis. 90% of European Caucasian mutations
Mutations tested by CFEU2?
50% mutations and uses capillary electrophoresis
What do Tube A and Tube B in CFEU2 contain?
Tube A- all mutation specific primers and WT F508. Primers for Poly T
Tube B- all other WT primers- only required to determine zygosity if mutation identified in Tube A and it is not F508
Dye coloures in CFEU2:
a) WT
b) mutant
c) hypervariable markers
a) green
b) blue
c) red- if these are absent the test has failed, can also use as an identity check
How is testing for rare CF mutations carried out?
Gene screen by means of sequecning, MLPA or linkage analysis (can put markers around cf gene to identify the high risk haplotype)
How could false negatives/heterozygotes being wrongly called as homozygotes result from eithr CF mutation assays?
snp under primer binding site
Mutation which is more prevalent in Asian populations? What is the issue with testing for this?
Y569D. Don’t get ethnicity details on referral cards and using surname is not an accurate method of determining ethnicity
What age does an individual need to be in order to be tested for CF?
at least 16
What is the risk of the asymptomatic sibling of a patient being a carrier?
2/3