Diagnosis And Treatment Of Cystic Fibrosis Biology Essay

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Cystic fibrosis is a chronic, autosomal recessive disease caused by a single gene mutation on chromosome seven. Genetic testing is a significant technique used to identify this faulty gene which will lead onto confirming whether or not the patient has Cystic Fibrosis; a useful diagnostic tool. Ideas are continuously being put forward to improve CFTR mutation. Gene therapy is a major strategy that is persistently being developed to produce a correct therapeutic response by normalising cellular function. Genetic therapy exploits the use of DNA and RNA. Oligonucleotides are used to inhibit the translation of the faulty mRNA which can stop protein biosynthesis. And the application of Adenoviruses used as vectors to transport new genetic material into human tissue.


Cystic fibrosis is a chronic [1], autosomal recessive disease caused by a single gene mutation on chromosome seven [2] (ref to fig. 1). Cystic Fibrosis transmembrane conductance regulator protein is affected by the ΔF508 mutation which deletes a codon on the CFTR gene [3]. It affects mostly the respiratory system characterised by inflammation of the airways leading to a reduced life expectancy [4]. And can also cause distress to the digestive system and the sweat glands [2]. This is because the ion transport in cells is defective causing viscous mucus to be produced [1] [2]. The diagnosis and the potential treatments for this disease are important to allow the sufferer to maintain a healthier and a much less sufferable lifestyle.

Fig. 1. The inheritance of autosomal recessive gene

If both parents are heterozygous, in that they carry one normal CFTR gene and one faulty CFTR gene, there is 1 in 4 chance of a child inheriting Cystic Fibrosis. So there is 25% chance for child to inherit 2 normal CFTR genes and no phenotypic characterisation of the disease. 50% chance of the children inheriting 1 faulty CFTR gene, so being carriers of the gene. These children will not show any characterisations of the disease because it is recessive and will be masked by the dominant allele. U.S. Department of Health & Human Services (2009); Example of an Inheritance Pattern for Cystic Fibrosis. Available http://www.nhlbi.nih.gov/health/dci/Diseases/cf/cf_causes.html [7th March 2011]


Genetic testing is a significant technique used to identify faulty genes which will lead onto confirming whether or not the patient has Cystic Fibrosis. However non genetic testing for the disease is just as important [5]. Sweat testing is an efficient way of testing the patient whether the symptoms they have is due to Cystic Fibrosis [5]. This is because in people suffering from this disease the amount of NaCl secreted in their sweat will be remarkably higher than the normal population, approximately 20 mEq/L more[5]. This is a much more rapid and noninvasive way of determining whether the disease is present in a suspected patient [5] and can be further verified by carrying out genetic testing techniques.

Genetic Confirmation of Diagnosis

As well as sweat testing, genetic testing is an efficient diagnosis used to confirm the presence of Cystic Fibrosis in a patient. A northern blotting technique is an efficient way of detecting mutation by examining mRNA present in defective cells. To analyse the mRNAs coding for CFTR protein, they are extracted from a sample of cells obtained from suspected Cystic Fibrosis patient [6]. They are then separated by size using gel electrophoresis and the fractioned RNA molecules are made available to DNA probes by Northern Blotting technique [6] ( ref to fig. 2). By using this procedure it is possible to verify the gene expression in cells and how it controls protein structure and function [6]. The hybrisided RNA molecules can be compared with RNA bands obtained from a known Cystic Fibrosis affected cell, used as a standard [6]. The size and the position of the bands are paralleled allowing the detection of the disease [6]. Northern blotting is beneficial due to the fact it can detect very minute changes in gene expression [6]. But can have problems such as contamination, where RNAses can breakdown RNA molecules very easily [6].

Fig. 2. Northern Blotting-the recognition of RNA molecules by gel transfer hybridisation.

(A)Gel electrophoresis is used to separate RNA molecules according to size [6]. (B) The gel obtained from electrophoresis is placed on filter paper that has been placed in a tray containing buffer solution [6]. (C) A nitrocellulose filter is laid on top of gel, along with a stack of paper towels and a weight. The separated RNA fragments on agrose gel will move onto nitrocellulose filter by capillary action [6]. (D) The filter is then removed cautiously of the gel and placed into a sealable plastic bag [6]. The bag contains a buffer solution with DNA probe that have been labelled by radioactive isotope or fluorescent tag. These hybridise with RNA in the right conditions [6]. (E) The filter is removed from bag and excess probes are washed off and the bands are visualised by auto photography [6]. Developmental Biology (1991); Developmental Northern Blotting. Available: http://8e.devbio.com/article.php?ch=4&id=32 [7th March 2011]


Gene therapy in humans is the insertion of modified genetic material into cells which will hopefully produce a correct therapeutic response by normalising cellular function [7] [8]. In theory the use of gene therapy is perfect for Cystic Fibrosis because of it being a single recessive gene mutation [8]. Furthermore taking advantage of the respiratory airways existing as an accessible surface to deliver the genetic material to [8]. However the introduction and expression of the newly given gene to produce a phenotypic correction in diseases such as Cystic Fibrosis is still vague [9]. Hence gene therapy is only used as a therapy not a cure.

DNA and RNA are mainly utilised in gene therapy [7]. Oligonucleotides are small single stranded DNA molecules that can selectively bind to mRNA to form a double stranded molecule [7]. This can inhibit the translation of the faulty of mRNA which can stop protein biosynthesis [7]. For therapeutic use in Cystic Fibrosis it can prevent the synthesis of the defective cAMP regulated chloride transporter, reducing the amount of viscous mucous produced [7]. By firstly inhibiting the synthesis of this protein in the apical membrane, viral vectors with correct recombinant DNA then can be used to deliver the new genetic material into defective cells [7] [8] [9].

Fig. 3. Recombination DNA

Restriction nucleases recognise specific DNA sequence on viral and human DNA [6]. The sequences are palindromic meaning that they have the same sequence in both 5' to 3' and 3' to 5' direction [6]. They cleave the DNA at these sites usually leaving sticky ends of exposed nucleotides [6]. Fragments of DNA from both human and viral sources can be joined together by complementary base pairing in a process called annealing [6]. DNA ligase is used to seal the nicks to form a continuous strand of DNA [6]. Restriction nucleases are naturally occurring enzymes found in bacteria that are used to breakdown viral DNA in a protecting mechanism [6].

Anon; s.I.; s.n. Available: http://www.obgynacademy.com/basicsciences/fetology/genetics/

Adenoviruses are non-enveloped icosahedral viruses which contain double stranded DNA [8]. It has a very small genome usually about 40 kilobases [8]. These viruses naturally infect human cells i.e. in the respiratory tract, so they are readily used in gene therapy to infect epithelial cells and transmit their modified DNA [7] [8] [9]. Recombinant DNA is made by using restriction enzymes to splice the CFTR gene into the viral DNA by cutting at the palindromic sites [6] (ref to fig. 3). The sticky ends of CFTR produced by restriction enzyme will be complementary to sticky ends in DNA of the virus causing them to bind with high affinity [6]. Usually the E1 region of viral DNA is replaced by desired gene which will prevent the adenovirus from replicating leading to the lysis of human cell [7] [8] [9]. As well as reducing the immune reaction that will occur in humans, by controlling the quantity of viral protein produced [7] [8] [9]. However, adenoviruses have low efficacy on respiratory cells because of the viral receptors being on the basolateral side, instead of the apical side where CFTR protein is found [7] [8] [9]. Increasing the dosage of the vector can produce an immune response which can render the adenovirus ineffective [7] [8] [9].

These CFTR encoding vectors are given in fluid form [7] [8] [9]. The nasal epithelium and trachea is initially targeted using nasal aerosol, than few days later the lower ends of respiratory tracts are treated using a bronchoscope [7] [8] [9]. The adenovirus must deliver its DNA genome into the nucleus of a host cell so it can successfully replicate [9] (ref to fig. 4). This is how the use of using recombinant viral DNA to infect human cells will work as a successful gene therapy [9]. The detection of mRNA expressing CFTR protein can be found in samples obtained from patients, and an increase in CFTR protein production in these cells lasted for a few weeks [7] [8] [9].

Fig. 4. Viral entry into host cell

a) Viruses attach to receptors on the host cells using membrane proteins this causes the merging of the two membranes. The capsids that surround the viral DNA are released into the cytoplasm and travel to the nucleus. The DNA is then releases in the nucleus via the nuclear pore. b) The human CFTR gene spliced into viral DNA can be transcribed into correct mRNA, which therefore is translated into working CFTR protein.

Donald M. Coen & Priscilla A. Schaffer (April 2003) Virus replication cycle; Nature Reviews Drug Discovery 2. Accessible: http://www.nature.com/nrd/journal/v2/n4/fig_tab/nrd1065_F1.html


In conclusion, the use of genetic strategies is a major advance in the diagnosis and therapy of a disease. It reinforces the information obtained from the patient such as the symptoms that occur and non-genetic testing for a confirmed diagnosis of the disease. But when it comes to potential therapies, there are many downfalls to the procedures due to variations in the human body such as immunity. Immune reactions against the vectors are a major problem, which could be possibly solved by using completely synthesised vectors. So whilst genetic strategies are very important when it comes to diagnosis, it probably should not be the only procedure used when it comes to therapy.