Basic Molecular Genetics Of Cystic Fibrosis Biology Essay
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Cystic fibrosis is a membrane transport disorder which is commonly affected in the autosomal recessive disease of white populations. It is also identified at birth by a certain condition called " meconium ileus" which means intestinal obstruction. In the advance stages, clinically it has been diagnosed by sticky viscous secretion of the pancreas and lungs.(Gelehrter,1998). Many years of direct research has failed to recognise the specific gene which is involved in cystic fibrosis (CF), although there are some various indirect research done which has indicated that there appeared an abnormality in the transport of ions during the process of cell membrane development. A very common diagnosis has been used to recognise this disease which is high increase of chloride content in sweat. Children suffering from this disorder develop pancreatic insufficiency which is usually treated with enzyme supplementation, antibiotics and physical treatments which are used to reduce the chest infection. Earlier the survival average is about 25 years. (Gelehrter,1990). But in a recent study , it has been said that the life expectancy for cystic fibrosis patients has increased over the last 40 years . It has been necessary to identify the respiratory viruses in Cf to make the clinical decision to proceed with the necessary treatment. (cited in Wat, 2008). It is said that one can calculate approximately the frequency of heterozygous carriers would be about 1 in 25 individuals. There are various research works done on cultured cells and animal models to further study on the basic defects of the disease and find a successive treatment. (Hodson,M.E., 1995).
From centuries the study and research of Cystic fibrosis has been done. Earlier people started diagnosing this condition in the newborns due to the salty taste in their skin. The survival rate was so low as six months. But as years followed , during the 1930s , a scientist named Guido fanconi had described the symstoms as gastrointestinal symdrone but latter named it as cystic fibrosis. The research had advanced when the cystic fibrosis gene was discovered following the study of mendel's law researched by Gregor Mendel. The genetic knowledge on this particular disease was made more improving which indirectly improved the survival ascepts of CF patients. By 1980s, a deeper understanding of human genetics were developed. In 1989, two researchers had isolated the damaged gene in causing CF which was the main cause in affecting the chloride channel function. These were the channels which were helping in the movement of water and salt in the cell walls. Further study on this is making it possible to understand and find a treatment for this genetic disease.
In recent years , various study via Gene theraphy has been helping various researches to fix the damaged gene. If this particular gene is fixed , the possible prevention, treatment and cure of CF would become a (Giddings,2009)
Basic molecular genetics of Cf:
In the year 1985, several groups were experimenting on gene therapy by linkage analysis, a linkage was observed on the chromosome 7 , which in turn proceeded with testing some of the markers on chromosome 7 where they found CF gene near to these markers (met and J3.11) . Techniques like physical mapping showed that the distance between these markers was approximately 1.6 million bp, which is as large for approximately 50 genes.Various interesting research were made in which it was found that a vast majority of chromosomes carrying the Cystic fibrosis mutation were also carrying a particular alleles for XV-2c and KM-19 which was further named as - allele for XV-2c and + allele for KM-19 . This type of hapotype (-/+) showed approximately in 25% of normal chromosomes , whereas rest showed other different patterns. A term was defined for this type of disease gene associated with a allele with the nearby markers , which was named as ' Linkage Disequilibrim' . This term was made used to understand that these CF mutated genes were being descended from a common ancester. (Gelehrter,1998)
In a recent research, a genetic analysis was conducted by carefully cloning of a 500,000 bp candidate which resulted in three possible genes from the specific region which was studied. In testing under northern blotting technique it was found that this specific gene was coding for a 1480- amino acid protein including 26 exons with a length of 250,000 bp of DNA and the mRNA transcript was found to be 6129 bp long. (http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cftHYPERLINK "http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cftr.shtml"rHYPERLINK "http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cftr.shtml".shtml)
In further studying a cloned and sequenced cDNA was prepared using the sweat glands of a Cf patient , it was found that there was a difference in exon 10 from a normal sequenced gene. There was a deletion in the 3 -bp found which might have caused the deletion of a single amino acid (phenylalanine) at the specificresidue 508. (Gelehrter,1990). This type of mutation is known as Delta F508 or F508. Patients with homologous delta F508 mutation will tend to suffer with severe symptons of this disease , causing a very heavy loss of chloride ion transport.This causes an improper balance in the sodium and chloride ion ratio , eventually secreting a thin , mucus secretion in the lung area which traps bacteria , this causes severe lung infection,resulting a high mortality rate in CF patients. (http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cftr.shtml)
This gene was initially cloned in 1989 in which it was mapped to human chromosome 7q21 , with more interest in this particular region, researches were able to indentify 2 more closely linked markers other than (labelled MET and D758) which were D75122 and D75340, those were 10 kb part. Using lambda genomic DNA libraries , 280 kb of DNA were sequenced which were used to make long range restriction maps. These were also probed with cDNA clones to isolate a total of 500 kb. This can be seen in the figure
By using various approaches like screening zoo blots, northern blots and cDNA libraries the genomic DNA clone was isolated. Four different regions were isolated , out of that , only one proved to be CF gene which was 6.5 kb sequence in length. Eventually , it was concluded that the CF gene controls the CFTR (cystic fibrosis transmembrane regulator) which encodes chloride channels which were responsible for the water transport on the plasma membrane of the epithelial cells which were connected to the lung airways . This mutation was causing the problem. (http://homepages.strath.ac.uk/~dfs99109/BB310/CFgene.html)
Structure of the CFTR gene:
After sequencing the CFTR gene , further study was done on it. The sequenced coding region of this gene showed a polypeptide molecular mass of 168138 daltons. The best site seen in this predicted protein was that it showed two repeated motifs, which showed some similarities to the nucleotide-binding domains (NBD) which is present in the membrane bounded protein. These membrane consisted in them six hydrophobic regions which composed 234 amino acids. This can be seen in the figure below
These 2 hydrophilic domains contains approximately 150 amino acids in which some phenylalanine residue has been deleted at the region of the first NBD which shares similar homologies with the proteins which binds with the nucleotide. These two motifs are connected by a highly charged cytoplasmic domains referred as R- domain or regulatory domains. This domains encodes 13 exons that help in the phosphorylationby protein kinase K and rest helps in binding sites by protein kinase C.
These domains and nucleotide binding folds in the binding sites suggest that hydrolysis is involved in the transportation of CFTR gene.
It was found out that CFTR gene had a addition promoter region with a size of 3.5 kb , later on, it led that these promoters showed the characteristics of 'housekeeping gene' along with tissue specific functions. There were five reasons that were focused to show that the CFTR gene had these charactertics which were as follows:
There were no TATA box element within the first 500 basepairs of the transcription sites.
The GC content in the promoter region was high.
There were addition multiple minor transcription sites identified along with the major transcription sites.
Couple of SP1 sites were also identified.
There was a very low expression of the CFTR gene found in the epithelium layer.
Another conclusion was made that the CFTR gene might control transcription regulation.
(prin mg , tho)
Phenotypic function of the diseased gene: To understand the phenotype of the disease , it has been confirmed that an individual suffering must have two copies of a mutated Cystic fibrosis transmembrane conductance regulator (CFTR) gene to be expressed. It has also been understood that the development of this disease in different individuals varies due to differential influence by environmental and genetic factors. This various different forms of other genes can affect the phenotypes of the mutated gene of this disease. (http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cftr.shtml)
As it been understood that the most common mutated gene found in most cystic fibrosis defective gene is the delta F508. A detailed study about this gene was done in which it was found that CFTR protein helps in encoding single amino acid at position 508 on the chromosome 7 . As this mutation affects the CFTR to perform its work in the cell and prevents it from locating itself in the cell membrane. Usually it is seen that, a newly synthesized CFTR protein adds itself to the missing chemical group by folding itself into appropriate shape which are escorted by their specific molecular chaperons to the surface of the cell. Because of the presence of this mutated CFTR gene , the imperfect CFTR binds onto the cell membrane which has a defect while opening and closing during regulating the chloride ion flow within the cell membrane. (http://www.healingwell.com/library/cysticfibrosis/info1.asp)
There are different mutated gene in which some do not this synthesis or any modification or integratation into the membrane of the cell. Sometimes, these mutated genes fail to even respond to the signals within the cell that are responsible for the opening and closing of chloride flow. In some cases, the CFTR protein enters the cell membrane and also responds to the cell membrane but there would show a problem when the channel opens and there is improper flow of chloride ion out of the cell. Different mutations vary with different patients, patients with absent or very low CFTR protein in its cell membrane would cause severe disease causing depleting pancreatic functions. There are patients which have delta F508 mutation on both CFTR gene copies , this causes very poor pancreatic function which would secret high amount of mucus causing varying degrees of infection in the lungs. Another CFTR mutation namely R117H also develops a partial functioning CFTR protein , this mutation pairs with the severe mutated gene delta F508 causing the CF disease severe but varying in lung disease. It has been seen that some men who have been detected with this particular mutated gene R117H shows only one symptom of CF that is being Infertile which is due to lack of vas deferens.( http://www.healingwell.com/library/cysticfibrosis/info1.asp)
Inheritence of the CF gene:
Diagnosing cystic fibrosis
In most cases, cystic fibrosis is diagnosed by screening tests, which are carried out very early in life. However, some babies, children and even young adults have some unexplained illness in future so they are diagnosed later. There are four main ways of diagnosing cystic fibrosis:
In newborn screening there are two tests the first one is blood test in which small amount of blood is taken from the baby's heel and transferred onto the card and it is examined in the laboratory for CF and it is also test inherited conditions, such as sickle cell anaemia and phenylketonuria.. Another test is genetic test in which saliva sample taken from the cheek and check whether a newborn has faulty CFTR genes.
This test is done during the women's pregnancy to know that fatus has CF or not. There are following
Amniocentesis: in it inserts a needle through abdominal wall into the uterus and take amniotic fluid surrounds the baby and tested in the laboratory
Chorionic villus sampling: In it needle passed through the stomach into the womb sometimes a fine tube passed through the vagina into the neck of the uterus as an alternative. And take chorionic tissue which is a tiny piece of the developing placenta and these chromosomes which are in the cells of the tissue are examined in the laboratory for CFTR genes are normal or not .
Both tests have risk of complication and also chance for miscarriage so it is offered only those mothers who have high risk of having child with the CF.
People who have two CFTR genes one is normal and another is faulty are CF carriers. So carriers pass faulty CFTR genes on to their children. This is simple test which is uses mouthwash to make sure that person is a carrier of cystic fibrosis. Swishing the mouthwash to collect the cells. And these containing cells are checked for the faulty gene.
If blood and genetic tests have positive sign for CF so for confirmation sweat test diagnosis using This test measure the amount of salt in sweat. For that in one treatment room small area of skin on an arm or leg tested. Rub this skin with sweat producing chemical and then produce gentle electrical current with help of electrode it gives warm feeling .collect sweat on a pad or paper for analze.High levels of salt confirm diagnosis of CF. (180,205)
This analysis is used after sweat test if little sweat measured in newborns. Blood samples are taken after 2-3 days of birth and examined for immunoreactive trypsinogen (IRT) which is the increased levels of pancreatic enzyme. If IRT level is high then also it is not strong indicator of CF. Because infants with intestinal malformations and condition as bowel obstruction also have high level of enzyme which is for function of impair pancreatic.
Lung X-rays and function tests:
In the tests measure the amount of air the lungs can hold and how fast a person can breathe out to show how lungs add oxygen and remove carbon dioxide from the blood with the help of chest X-ray which show lung scarring from recurrent inflammation.This test do not confirm a diagnosis of CF but help in evaluating the severity of an individual's lung damage.
This test give signs of sinusitis, which indicates an infection and inflammation of nasal cavities. The X-ray shows the underlying cause becomes important for sinusitis. By cause of sinusitis is showing allergies and poorly functioning immune system.
DNA mutation analysis:
For highly specific result we used this technique for CF to look for the presence of many mutations in the CFTR gene. In it DNA isolated and purified from the blood sample or from a smear inside the cheek and for detect disease genes and indentify mutation within a specific DNA sequenced used micro array technology
For this test, take a sample of your sputum (spit) to see what bacteria are growing in it. If there are bacteria called mucoid Pseudomonas, then it is advanced CF that needs aggressive treatment. (Giddings, 2009)
In early years, there were no proper complete cure for cystic fibrosis . This has changed in recent years due to various researches done to treat CF which have been improved drastically. The aim for the treatment of cystic fibrosis are to Prevent infection in lungs,Remove the thick and sticky mucus from lungs, Prevent blockages in intestines and to Provide adequate nutrition.(http://www.nhlbi.nihHYPERLINK "http://www.nhlbi.nih.gov/".gov/)
Treatment for lung problems :
It is essential to begin the treatment by reducing the infection and removing the thick and sticky mucus from the lungs. This can be done by performing the following lung treatment methods.
CHEST PHYSICAL THERAPHY(CPT):
This theraphy is also called percussion. CPT include pounding the chest and back over and over with the hands and a device that reduce the mucus layer from the lungs. Breathing techniques are also helpful to remove mucus from lungs. This techniques involve forcing out of some short or deeper breaths and following with a relaxed breath. The major purpose of this therapy for CF is to increase clearance of mucus and control infection in the lung and also to provide adequate nutrition and prevent intestinal blockage.(http://www.nhlbi.nih.gov/).
This is the primary treatment for the cystic fibrosis. Using medicines it helps in treating lung infection, decreases the swelling ,reduces the mucus and also opens the airways. The following types are the antibiotics used for cystic fibrosis treatment:
Oral antibiotics are use to treat the lung infection.
Inhaled antibiotics are used to control the infection caused by the pseudomonas bacteria.
Intravenous medicines are used for control the severe infection.
Anti-inflammatory medicines used to reduce swelling in airways.(http://cysticfibrosis.emedtv.com/).
Drugs or steroids such as azithromycin, ibuprofen, prednisone and pentoxifylline are used to reduce inflammation.
For the treatment of bacterial infection various lung antibiotics such as nebcin, tobrex, ciprofloxin, myambutol,biaxin, azactam are used(.http://wwHYPERLINK "http://www.healthscout.com/"w.healthscout.com/).
Exercise: Aerobic exercise can help to reduce and clear the mucus from the airways. Regular exercise also enables to cut back on the CPT.
Intracytoplasmic sperm injection is used to provide fertility for men with cystic fibrosis
Lung transplantion is a effective therapeutic option for the cystic-fibrosis patient. The successfully lung transplanted patients are able to control diabetic problems easily as the patients exercise capacity improves and the respiratory infection reduces.(Madden,1992).
In lung transplant ,the infected lung is replaced with a healthy lung by surgery. If CF is very advanced then lung transplantion is a good option. It is a major operation and sometimes also leads to some serious complications (http:HYPERLINK "http://www.mayoclinic.com/"//www.mayoclinic.com/).
If oxygen level is very low in the blood then oxygen therapy is very effective(http://cystic-fibrosis.emedtv.com).
C.F causes many digestive problems such as slow growth, pain, intestinal gas and severe. Nutritional treatment can improve the growth, development and strength of patient. It involves a balanced diet with low fat and high protein which make strong the patient to resist aganist some infection (http://www.healthscout.com). It contain pancreatic enzymes to digest protein and fat and absorbance of various vitamins. Nutriational therapy also contain supplements of different vitamins. The other treatment for digestive problems is medications for the treatment of intestinal blockages (http://www.nhlbi.nih.gov/).
Gene therapy involve transfer of DNA for therapeutic purpose. For the treatment of cystic fibrosis two approaches have been used. In the first approach, adenovirus was used as a vector, it has the epithelial cell lining. In this approach, adenovirus type-2 late promoter drives the CFTR gene. In the second approach, positively charged liposomes was used which can bind negatively charged DNA on its surface. These initial experiments have been used to change the electrical conductance of the nasal membrane. By these approaches a little correction of conductance abnormalities is possible. In the recent studies aerosol inhalers is used to saturate the upper part of the respiratory system. The reversal of the path physiology is possible if the correct form of CFTR gene is expressed.
At present, retroviral-mediated gene transfer system is used for gene transfer for gene therapy of CF. Adenovirus is a suitable vector for gene transfer to the airway because it uses humans as a permissive host and it has a natural tropism for the lung .Liposome-mediated DNA transfer system is also used for gene transfer in gene therapy. Liposome is positively charged interact with DNA which is negatively charged and form lipid-coated DNA complex Different positively charged liposomes used to transfer the CFTR including DOTAP, DMRIE and DOTMA-DOPE.
Several new treatments are developed to treat the CF. In one case, the sodium-channel inhibitor amiloride is insert to the airways of the patients which inhibit sodium absorption. The other possible approach is to allow to bypass the CFTR chloride channel and regulate other chloride channel present in the membrane of airways. The most effective way to treat the CF is to insert a new and normal copy of CFTR gene into the airway of respiratory tract of CF patients.
In recent years , various research works have improved the way to diagnose and treat cystic fibrosis. In the year 2006, it was published in the 'Science daily' that certain scientists from Stanford University, USA were able to find out the basic defect causing CF. In which they had concluded that the airway blockage caused in CF was not caused due excessive fluid being reabsorbed within the glands but due to lack of fluid secretion from cystic fibrosis glands. (http://www.sciencedaily.com/releases/2006/03/060318232035.htm)
In another research work done in the year 2008, certain scientist from USA were able to discover that a particular protein called CFTR were either missing or not present in the lungs of the CF patients which helps in fighting various bacteria like Pseudomonas aeruginosa when inhaled. Further studies are conducted to find the treatment through these proteins. (http://www.sciencedaily.com/releases/2008/09/080910210525.htm).
In 2010, A team led by Professor Justin Hanes from John Hopkins university had researched on a mucus penetrating biodegradable nanoparticles which would help in delivery wide range of therapeutic molecules from small to various size of macromolecules to reduce the infection of lungs of the CF patients.
In another research study done this year 2010, a team of researches were able to find out that a certain calcium- activated chloride channel were able to enable CF mucous cells to secrete normally which is required in the CFTR channel. So, it was concluded that drugs should be produces to enchance the ability of the calcium response which helps in activating the calcium activated chloride channels which are secreted in the CF cells. (http://www.sciencedaily.com/releases/2010/09/100917151848.htm)
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