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Cystic fibrosis: molecular genetics and pathophysiology 1. & 2. Classify the common mutations which are found in CF and Explain how different mutations result in differing severity of clinical disease, according to whether cAMP stimulated chloride secretion is reduced or absent. Cystic fibrosis (CF), or Mucoviscidosis, is a disorder of epithelial transport affecting fluid secretion in exocrine glands as well as the epithelial lining of the respiratory, gastrointestinal and reproductive tracts. As a result, the disorder leads to abnormally viscous mucus secretion, which causes most of the clinical features associated with CF ie. - Recurrent pulmonary infections leading to chronic lung disease - pancreatic insufficiency - steatorrhea - malnutrition - hepatic cirrhosis - intetinal obstruction - male infertility CF is an Autosomal Recessive disease and it is the most common lethal genetic disease in Caucasian populations (1 in 2500 births). The gene responsible for CF codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP regulated Cl-ion channel found in the apical membranes of secretory epithelial cells. The protein encoded by CFTR has two transmembrane domains, two cytoplasmic Nucleotide Binding Domains (NBD) and a Regulatory Domain (R Domain). Activation of the protein is as follows; Agonists, such as acetylcholine, bind to epithelial cells, resulting in the increase of the second messenger Cyclic Adenosine Mononphosphate (cAMP). cAMP activates Protein Kinase A (PKA) The R Domain contains phosphphorylation sites, which are subsequently phosphorylated by PKA This causes a conformational change allowing the binding of ATP at the NBD This also cause conformational change allowing the opening of the two transmembrane proteins through which chloride can now pass Hydrolysis, or removal of ATP causes channels to close Pathophysiology Although initially characterised as a Chloride-conductance channel, it is now recognised that CFTR can regulate multiple ion channels and cellular processes. These include inward potassium (Kir6.1), gap junction and epithelial sodium (ENaC) channels. Of these, the most pathophysiologically relevant is the interaction of CFTR and ENaC. It is important to note however that the functions of CFTR are tissue specific; meaning the impact of a mutation is also tissue specific. ENaC is situated on the apical (luminal) surface of the exocrine epithelial cells, and is responsible for intracytoplasmic sodium transport from luminal fluid, rendering it hypotonic. It is important to note that ENaC is inhibited, or has reduced activity by normally functioning CFTR. Thus, in cystic fibrosis, ENaC activity increases, as does sodium transport into cell. This phenomenon has important consequences for respiratory and gastrointestinal pathology. Human sweat ducts are an exception to this however, as ENaC activity decreases as a result of CFTR mutations, resulting in hypertonic luminal fluid, high in sodium and chloride. In the respiratory and GI epithelium, the CFTR channel is one of the most important avenues for luminal secretion of chloride. A mutation or abnormality in the CFTR not only decreases chloride secretion, but it also results in increase sodium reabsorption due to lost ENaC inhibition. Both of these ion disturbances result in passive water reabsorption from the lumen, lowering the water content of the surface fluid layer coating the mucosal cells. In the lungs, this dehydration leads to defective mucocilliary action (can’t beat properly) resulting in the hyper-concentrated viscous secretions that can obstruct the air passages and predispose to pulmonary infections. CFTR also mediates the transport of bicarbonate ions. Normal tissue cells secrete alkaline fluids, whereas as those with defective CFTR secrete acidic fluids (as no bicarbonate). Decreased luminal pH can have various adverse effects, such as mucin precipitation and plugging of ducts (Pancreatic insufficiency). Mutations Since the CFTR gene was discovered, more than 800 disease causing mutations have been identified. These mutations can be grouped into five classes based upon their effect on the CFTR; Class I: Defective Protein Synthesis – mutations cause a complete lack of CFTR protein at apical surface of epithelial cells Class II: Abnormal Protein folding, processing, and trafficking – mutations result in defective processing from the Endoplasmic reticulum to the Golgi apparatus – protein does not fold properly and become glycosylated, instead is degraded before it reaches the surface – total lack of CFTR at epithelial surface Most common CF gene abnormality is a Class II mutation that leads to the deletion of three nucleotides coding for phenylalanine at amino acid position 508 (∆F508). Found in approximately 70% of all CF patients worldwide Class III: Defective regulation – mutations prevent activation of CFTR by preventing APT binding and hydrolysis, the pre-requisite for opening and closing. Thus normal amount of CFTR, but it is non-functional Class IV: Decreased Conductance – Mutations to transmembrane domains of CFTR that form the pore for chloride transport. Normal amount of CFTR at apical surface, but with reduced function. Class V: Reduced Abundance – mutations affecting intron splice sites of CFTR promoter – resulting in reduced amount of protein As CF is an autosomal recessive disease, affected individuals harbour mutations on both alleles. However, the combination of mutations on the two alleles can have a major effect on the overall phenotype. Two ‘severe’ mutations, such as those of class I, II and II, produce a virtual absence of membrane CFTR and is associated with the Classic CF Phenotype; pancreatic insufficiency, sinopulmonary and gastrointestinal symptoms. The presence of ‘mild’, class IV or V mutations, on one or both of the alleles results in a less sever phenotype. This is deemed nonclassic or atypical CF. 3. Outline the current hypothesis for the prevalence of the CF gene. As already mentioned, Cystic fibrosis has simple Mendelian autosomal recessive inheritance. Affected individuals will have two copies of the mutated CFTR gene, one inherited from each parent; carriers will have one normal and one mutated CFTR gene and their health will not be affected. However carriers will have the potential to pass on the gene to their offspring. Brothers and sisters of affected individuals are at increased risk (1 in 4) of having CF because both parents will be carriers. Cystic fibrosis remains one of the commonest life threatening autosomal recessive condition affecting Caucasians. The incidence is about 1 / 2,500 live births and the carrier frequency is 1/25. It is uncommon in Asians and Africans. The high prevalence of the CF gene in certain populations has led to speculations that there may be some heterozygote advantages. It has been postulated that carrier status may be linked to improved survival (less chloride loss with the diarrhoea) following infections such as cholera and typhoid. 4. Describe the cellular and molecular basis for strategies for gene therapy Gene therapy can be broadly defined as a treatment involving transfer of genetic material into the genome of a patient with resulting therapeutic benefit to the patient, Two General Strategies are being developed for the treatment of disease based on somatic gene therapy: Ex vivo gene therapy- involves transplantation of genetically modified autologous cells In vivo gene therapy- involves directly delivering the gene to a specific location in the patient’s body In principle, CF and other single gene disorders should be amendable via gene therapy. In vitro it has been possible to correct the chloride defect in epithelial cells of CF patients, with a single copy of the wild-type CFTR gene being able to revert the CF phenotype. Both the identification of the CFTR gene in the lungs, as well as the use of Viral and Non-Viral vectors, provides a possible source of treatment for CF sufferers, however clinical trials are still in the early stages.