Molecular Diagnosis of

Cystic Fibrosis
About Cystic Fibrosis

• Autosomal recessive disorder caused by

mutations in the gene for the protein cystic
fibrosis transmembrane conductance
regulator (CFTR)

• Prevalent in European Caucasians (1 in 29

Europeans and Americans)

• The average life expectancy is between 37

and 50 years in the developed world.
Cystic fibrosis transmembrane conductance regulator
CFTR is positioned on chromosome 7q31.2 . CFTR contains 27 coding exons; genomic sequence is ~230 kb;
mRNA is ~6.5 kb.

CFTR is 1480 amino acids with a mass of ~170,000 daltons. CFTR is in the ATP-binding cassette family of
transporter proteins. The CFTR protein contains five domains including two membrane-spanning domains,
a regulatory domain, and two nucleotide-binding domains that interact with ATP.
Functions of the CFTR protein
• The CFTR protein is a membrane ion channel that
transports Cl– and HCO3– ions bi-directionally

• CFTR channels are found in the apical membrane

of epithelial cells throughout the body

• Apical side - Inner side of ducts of various glands

• Proper functioning is required for the ionic

balance and hydration of secretions in organs
including the lung, liver, pancreas, digestive tract,
reproductive tract and skin.
Pathophysiology of Cystic Fibrosis
CFTR Gene defect

Defective ion transport

Lungs
Sweat gland
Dehydration of viscous secretions
Liver
Pancreas
Defective clearance of secretions GI tract

Duct obstruction Reproductive system

• Mutations in the CFTR gene can affect the quantity and/or the function of
CFTR channels at the cell surface.
Infection Inflammation
• The type of mutation has a significant impact on the extent of abnormal
chloride transport and, consequently, on the patient’s symptoms.
CFTR mutations
• There are currently 2005 mutations listed in this CFTR mutation database.

• Six classes of CFTR mutations :

 Class I mutations lead to defective protein products

 Class II mutations result in defective protein processing

 Class III mutations have a defect in the channel regulation

 Class IV mutations are defective in conductance through the channel and represent milder mutations

 Class V mutations results from abnormal splicing and cause reduced synthesis

 Class VI mutations increase the turnover rate of CFTR protein at cell surface

• The type and frequency of mutations is very variable from ethnic population to population

• The most common mutation, F508del, reaches frequencies of about 70% in Northern European populations.
CFTR mutations – Class I
CFTR mutations – Class II
CFTR mutations – Class III
CFTR mutations – Class IV
CFTR mutations – Class V
CFTR mutations – Class VI
CFTR mutations
Approximate Common CFTR Associated
Predominant CFTR Protein Functional
Class Worldwide Representative Domain Phenotype
Mutation Type Outcome Consequence
Frequency Mutations Location Severity

∼10%
G542X, W1282X, NBD1, NBD2,
I (Ashkenazi, Nonsense, splice High No CFTR No Cl- transport
621+1G→T MSD1
50%)

Defective
II 70% Missense F508del, N1303K NBD1, NBD2 High No Cl- transport
processing

Defective
III 2%-3% Missense G551D, R560T NBD1, NBD1 High No Cl- transport
regulation

Minimal
Uncertain, < MSD1, Altered
IV Missense R117H, R347P Reduced expression &Cl-
2% MSD1 conductance
transport

Reduced
Uncertain, < Reduced
V Missense, splice 3349+10kbC→G Intron Reduced expression &Cl-
1% synthesis
transport
Diagnostic testing
The ‘standard’ and internationally accepted test for CF is the sweat test. However, with over 2500 CFTR mutations giving
rise to a wide range of clinical symptoms, there is increasing use of genetic analysis in the diagnosis and screening of CF.
Thus, both genetic and physiological tests are included in current diagnostic algorithms for CF.

CF diagnosis:
• Two abnormal sweat chloride values (≥60 mmol/L); and/or
• Presence of two disease-causing mutations in the CFTR gene

Testing is recommended in:

• Typical (chronic lung infection) and atypical (residual functioning of pancreas) presentation of CF symptoms
• CFTR related disorders like congenital bilateral absence of the vas deferens (CBAVD)
• prenatal and newborn diagnosis
• carrier testing in individuals with or without family history

Testing cascade:
• Search for most frequent mutations (ethnicity of population should be taken into account)
• Search for rare mutations
• Screen for unknown mutations
• Additional physiological tests - immunoreactive trypsinogen, nasal potential difference, intestinal current measurement
Frequently used tests for mutation screening
Amplification Refractory Mutation system – PCR:

• Two complementary reactions (two tubes) and utilizes 3

primers

• One primer is constant and complementary to the template in

both reactions

• Other primers differ at their 3' terminal residues and are

specific to either the wild type DNA sequence or the mutated
sequence at a given base - only one of these primers is used
per tube

• If the sample is homozygous mutant or homozygous wild type

amplification will only occur in only one of the tubes

• If the sample is heterozygous amplification will be seen in

both tubes
Frequently used tests for mutation screening

Reverse hybridization or Reverse dot

blot:

• Utilizes Allele-specific oligonucleotide

(ASO) probes, complementary to
mutant and normal DNA sequences, are
bound to membranes (dots or slots)

• DNA that has been amplified in

multiplex reaction(s) with biotinylated
primers is hybridized to the immobilized
oligonucleotides on the membrane

• Hybridisation is detected by adding

streptavidin-horseradish peroxidase.
Frequently used tests for mutation screening

Multiplexing is possible by using

different allele-specific probes
specific for particular mutations
Frequently used tests for mutation screening
Oligonucleotide Ligation Assay (OLA):

• Utilizes a common probe with fluorescent dye and two (or

more) allele specific probes with different 5’ tail lengths

• A pair of oligonucleotides designed to anneal to adjacent

sequences within a PCR product

• If perfectly hybridized, they are joined by a DNA ligase.

• Allele-specific probes are differentially labeled and the

products are identified by a computer software

• Multiplexed using different dyes and probes with different

length
Frequently used tests for mutation screening
Frequently used tests for mutation screening

Restriction fragment length polymorphism is also used but less frequently. It is now
replaced by other techniques and is considered obsolete

The above mentioned methods are screening methods, that is, they screen only for known
mutations

If screening tests fail to identify mutations in symptomatic CF, then unknown mutations can
be scanned
• By exploiting differences in hetroduplex mobility – Temperature gradient gel
electrophoresis (TGGE) , Denaturating high performance liquid chromatography (DHPLC)
and Single-Strand Conformational Polymorphism (SSCP)
• By direct sequencing
Scanning methods for mutations
Heteroduplex migration analysis:
• Differential migration of DNA heteroduplexes (2 mismatched single strands)
compared to homoduplexes run on a polyacrylamide gel.

• For individuals heterozygous for mutations – the mutant strand can combine with
strand from wild-type allele to form the heteroduplex

• In the case of homozygous mutant alleles - PCR products from a suspected carrier
are denatured allowed to reanneal to a normal PCR product
Scanning methods for mutations
Scanning methods for mutations
Temperature gradient gel electrophoresis (DGGE) and
Denaturating high performance liquid chromatography
(DHPLC) :

• based on the principle that partially denatured DNA is more

restricted and travels slower in a porous material such as a gel

• Facilitates separation of DNA based on melting temperature

which in turn is based on DNA sequence.

• Query and reference sequences are amplified by PCR,

denatured and renatured and then subjected to either TGGE or
DHPLC

• differential separation of mismatched heteroduplexes which

form after the reannealing of normal and mutant DNA strands -
detects heteroduplexes owing to their abnormal denaturing
profiles
Scanning methods for mutations

Single-Strand Conformational Polymorphism (SSCP):

• Screening for the presence or absence of mutation in an amplified exon.

• Basis: single DNA strand folds to form a complex 3-D structure maintained by
intra- molecular bonds.
• Presence of mutations variable folding variable electrophoretic mobility.
• Differential mobility between test and control DNA samples +ve mutation
sequencing.
References

• Cuppens, H., et al. (2004). Cystic Fibrosis. Molecular Diagnosis of Genetic Diseases. R.
Elles and R. Mountford, Humana Press: 221-244.
• Davis PB. Am J Respir Crit Care Med 2006;173:475-82
• Dequeker E, Stuhrmann M, Morris MA, et al. European Journal of Human Genetics.
2009;17(1):51-65. doi:10.1038/ejhg.2008.136.
• Eshaque B, Dixon B. Biotechnol Adv., 2006 24(1), 86-93
• Eaker S et al., Biosensors and Bioelectronics, 2005, 21(6), 933-939
• MacDonald KD et al. Paediatr Drugs 2007;9:1–10
• Row S et al. N Engl J Med 2005;352:1992–2001
• Welsh MJ et al. Cystic fibrosis. Valle D et al (Eds). OMMBID. The McGraw-Hill Companies
Inc. Part 21, chap 201, 2004