CYSTIC FIBROSIS: An overview

Cystic Fibrosis results from an autosomal recessive mutation on chromosome 7 that took 10 years to pinpoint, that is to say that it is not located on the X and Y sex chromosomes, and two faulty copies of the gene are usually necessary for the disease to manifest itself. Cystic Fibrosis, or CF for short, is the most common lethal inherited disease in the Western world, and around 70% of CF cases result from the deletion of a single amino acid from position 508 in the protein chain that is encoded by the normal gene. It is believed that 1 in 2000 children carry two defective copies of the CF gene in the Caucasian population. One in 25 people in Britain carry one faulty copy of the gene. Two such carriers have a 1 in 4 chance of having a child with CF and 1 in 2 chance of having a child who is a carrier. There are an estimated 2 million carriers in GB and 8 million in the US. The expected life span of a CF homozygote (two copies with mainfestations of disease) has now increased to over 30 due to improved health care and medication.

I shall briefly discuss the TISSUES THAT CF AFFECTS IN THE BODY, THE SYMPTOMS that are ASSOCIATED WITH CF, HOW WE DISCOVERED IT, WHAT THERAPIES CAN BE DESIGNED TO TREAT CF, HOW WE CAN DETECT THE FAULTY GENE IN THE POPULATION, WHAT THERAPEUTIC STRATEGIES ARE AVAILABLE and finally THE SOCIAL DILEMMAS THAT SCREENING FOR CF presents.

What tissues does CF affect in the body?

CF creates problems primarily for the body's single cell layers (epithelia) that line the lungs, intestines, pancreas, nasal epithelial lining, sweat glands and reproductive organs, known collectively as epithelia. The principal function of epithelia is to transport nutrients, salt and water between body compartments. In the 1950s CF was recognised by di'sant Agnese to be associated with abnormal salt transport in the sweat glands, but it was not until 1983 that Paul Quinton classically discovered that a deficit in Cl- secretion was responsible for this abnormal salt transport.

What are the symptoms associated with CF?

In the CF lung mucus that captures bacteria is thick and poorly hydrated, and hence and cannot cleared by the cilia leading to a `crackling cough' and opportunitstic infections that destroy the lung tissue, the primary cause of morbidity in CF patients. In the intestine and pancreas the deficit in the secretion of salt and water, hormones and enzymes leads to the improper digestion and absorption of nutrients, resulting in poor growth in affected children and infections of the intestinal tract. The CF defect in the reproductive tracts renders all CF sufferers infertile, and even males carrying only one affected copy of the gene have reduced fertility due to an obstruction of the vas deferens.

How the CF defect was discovered

In 1983 Paul Quinton elegantly demonstrated that reabsorptive sweat duct cells have an abnormally low Cl- ion permeability, in other words the cell membrane does not allow Cl- ions to cross readily, explaining why CF patients have increased concentrations of NaCl in their sweat due to decreased reabsorption. It has since been widely demonstrated in other fluid and salt secreting epithelia affected in CF that there is a deficit in the Cl- ion permeability that is stimulated by hormones that increase the levels of an intracellular messenger called cyclic AMP. To understand this defect we must first understand how epithelia transport salt and water.

Epithelial cells form a single layer joined together by tight junctions that separate the membrane into two domains, an apical one facing the duct or lumen, and a basal one facing the cellular tissue which is bathed by small blood vessels called the serosal side. Na+, Cl- and K+ ions are taken up by a cotransport mechanism in the basal membrane and the K+ and Na+ ions are actively recycled across the basal membrane by energy-dependent transport mechanisms, so that concentrations of K+ and Cl- are maintained within the cell that are higher than would be predicted if they distributed themselves freely across the membrane. When hormones interact with membrane located receptors they stimulate an increase in the levels of second messengers such as cAMP or Ca2+ in the cell. These second messengers activate channels that increase the rate at which K+ ions leave the cell across the basal membrane and Cl- ions leave the cell via channels that are present in the apical membrane. This results in a transepithelial potential gradient being established across the epithelial cell layer because of the negative charge carried into the lumen by Cl- ions. Positively charged Na+ ions are obliged to follow the negative charge gradient across the epithelial cell layer and water follows driven by the osmotic gradient. In CF Cl- channels normally activated by cAMP are not present or function abnormally, leading to an inability of the epithelium to secrete salt and water.

How do hormones that act from outside the cell increase the levels of second messengers inside the cell that regulate these ion channels? Hormones bind to receptor proteins located in the cell membrane and in doing so change their structure transducing a signal across the cell membrane. So-called G-proteins sense these structural changes in the receptor protein and break up into parts, one of which activates another enzyme called adenylate cyclase that converts ATP, the cell's energy currency, into cAMP, a reaction that is driven by the energy stored within the high energy phosphate bond of ATP. The cAMP molecules bind to regulatory subunits of a protein called protein kinase A causing it to release active protein kinase subunits. Kinases are enzymes that split ATP into ADP and a phosphate ion which is highly negatively charged and add this phosphate group onto a protein, changing its structure and thereby its function. In the case of ion channels, phosphorylation temporarily alters their structure and determines whether they are open or closed to the movement of ions.

How can we tell whether ion channels are open or closed in the cell membrane? In 1976 Sakmann and Neher developed the patch-clamp technique for which they were rewarded with a Nobel prize in 1991. Isolated cells can be approached with a microelectrode which forms a high resistance electrical seal with the cell membrane, allowing currents across the isolated patch of cell membrane to be measured. The currents across the membrane patch appear to show distinct openings and closings, and thus changes in the open state of the channel in response to signalling molecules like protein kinase A can be measured. If the isolated membrane patch is broken by suction then dyes, proteins or messenger RNA may be introduced into the cell interior. In the case of mRNA that encodes channel proteins injected into frog eggs, new channel proteins may be produced which allows ion channels to be studied in a cell-type in which they are not normally found.

Ion channels can either be studied individually or collectively in the intact cell membrane by a patch-clamp recording wherein the membrane patch has been perforated with the polyene antibiotic nystatin, allowing ion currents across the whole-cell membrane to be measured. Hormones that increase cAMP levels activate Cl- channels making the potential across the cell membrane more positive whilst hormones that increase the concentration of intracellular Ca2+ activate K+ channels. The CF Cl- channel is synergistically activated by protein kinases A and C, wherein a bigger response is seen in response to both hormones acting in conjunction.

In 1988 Mike Gray and co-workers classically showed that PKA activates small Cl- channels in the apical membrane of the pancreatic duct cell and the activation of small conductance Cl- channel by PKA in intestinal cells is rate-limiting in the coupling of a hormonal or nervous stimulus to the secretion of fluid and electrolytes. Channel activity in patches of membrane that are removed from the cell are only seen when the active subunit of PKA is applied. This occurred 1 year B.C., or before the successful cloning of the CF gene, or Cystic Fibrosis Transmembrane conductance Regulator, was announced in 1989 by Francis Collins and his team. After the gene had been isolated and sequenced, normal CFTR DNA was introduced by Kartner and Riordan into insect cells which do not normally produce this protein. In cells that did not receive the CFTR DNA no membrane currents were activated by cAMP, but in insect cells that received the CFTR DNA Cl- currents were activated, demonstrating that the CFTR was a Cl- channel. The structure of the protein encoded by the CFTR gene was determined and it turned out to be a member of a well-known family of ATP binding cassette (ABC) transporters which span the cell membrane. The R domain of the protein is phosphorylated by protein kinases and is believed to open like a molecular flap allowing Cl- ions to pass through.

Now that researchers knew that CF was due to a Cl- channel defect they have tried two strategies to correct the disease, one of these has been to use gene therapy to introduce a good copy of the CFTR gene into the CF lung either with a vaccinia virus or with membrane microspheres, called liposomes. The defect in genetically engineered CF mice has been successfully corrected with liposomes carrying the correct copy of the DNA. An alternative strategy is to activate other Cl- channels in the apical membrane. Boucher and Stutts showed that extracellular ATP acting as a hormone activates Cl- channels in CF lung tissue thus bypassing the CF deficit. Soon after the crypt regions of the small and large intestines were identified as the regions of the intestinal epithelium affected in Cystic Fibrosis (Walters et al., 1992; Trezise & Buchwald, 1992). Finally all the epithelia affected in CF had been identified and made accessible to further study.

How do we detect the CF mutation?

Mutations that are present near to the CFTR gene are inherited with the gene, so-called Restriction Fragment Length Polymorphisms (RFLP's), which mean that when the DNA in the region of the gene is cut up by enzymes that recognise specific sequences in the DNA the lengths of the fragments produced varies with the CF mutation. This can be detected when the DNA fragments produced are run through gels using an electrical field. Commercial tests are now available to screen individuals for the more common CF mutations, of which more than 140 have now been identified.

Strategies for overcoming the CF deficit

Having established that many mutations may lead to mild or severe Cystic Fibrosis, and that a deficit in the capacity to secrete chloride ions is predominantly responsible, the question remains how best to repair the deficit. Whilst researchers in the 80's and early 90's dreamed that drugs might be used to bypass the deficit, the last decade has seen the explosive advent of gene therapy, driven in all fields by the ground-breaking work of the CF teams. However, major barriers remain to the sucessful use of gene therapy in the treatment of CF as discussed below.

The first problem is that not all the affected cells take up the recombinant DNA that contains the 'good' version of the CF gene, in fact transfection efficiencies may be as low as 4% per nebulized spray. Clearly most or all the cells should preferably take up the reforming DNA. This is clearly not merely a problem of distribution in the lung, as in the early 90's we found transfection efficiencies of culture cells with a genetically modified vaccinia virus and lipofectamine were as low as 10%. Ten years later using lipofectamine and plasmids (circular DNA containing the gene to be transferred), our efficiencies had not improved. When cell cultures were treated with fluorescently labelled antisense DNA, 10-20% of cells took up the alien, naked DNA in abundance and 80% or more did not. I am beginning to think that this is because cells are only receptive to taking up foreign DNA at a certain stage of the cell cycle, possibly mitosis (division) and hence if only a certain percentage of cells in the airway are in a given stage of the cell cycle when treated, efficiencies will be low.

The second problem is one of turnover. The cells of the airway and intestines are constantly being shed and passed out of the system as we cough or defecate. Hence within days those cells which have been successfully transfected with the good DNA, as indeed some cells are, will be lost with the processes of wear and tear and renewal. Hence any gene therapy may have to be almost daily to succeed, and the toxicological consequences of daily high dose gene therapy are not known.

The third major problem is that when we introduce foreign DNA into whole organisms we are in a risky and uncharted territory. When cells detect single stranded DNA or RNA or are transfected with a virus, they recognize the DNA or RNA as foreign and enter into programmed cell death (apoptosis). Whilst this is not a bad thing in gene therapy within the realm of cancer, it is not a desirable feature in the treatment of CF. Further, although people have proposed introducing modified and weakened viruses to treat AIDS and other diseases through immunization, the same concerns regarding the use of genetically modified viruses apply. This is to say that even mild strains of a virus may have unpredictable consequences for human health. How is not clear. They may for instance cause new disorders, by oversensitising the immune system, inducing hidden viruses within the genome to replicate (multiply), or else to combine in new and unforeseen ways with other viruses. In effect we may be accelerating the evolution of viruses by the genetic recombination of new viral strains at a rate that does not take place in the natural world. A single laboratory can create thousands of new viral sequences every year by cutting, pasting, recombining and mutating existing ones. Whilst stringent safeguards do exist for their production and dissemination, the consequences of introducing any new strain into the human body cannot be entirely foreseen, and one or two deaths have already occured in response to gene therapy trials (as with most new drugs).

So where does this leave us? Certainly gene therapy and cloning are still in their infancy, and will almost certainly prevail through the irresistable forces of human will, ingenuity and seemingly limitless resources. For the next ten to twenty years however we should perhaps pay attention to Dr.Stutts who showed us that UTP, in combination with amiloride, can bypass the CF deficit through the activation of an alternate Cl- conductance in the airway. Perhaps other chloride conductances exist within the pancreas and intestine, and these too will provide viable treatment strategies in the shorter term. So for the next twenty years pharmacology will remain our primary weapon in the treatment of CF, that is as soon as we return our attentions to hunting for alternative Cl- conductances in these epithelia and determining how they operate.

The social dilemmas of screening for CF

Screening presents ethical dilemmas not only for the medical profession but also for society. Such tests do not reveal the carriers of the remaining 15% of rare CF mutations, and therefore testing cannot tell people for sure that they are not carriers. A study of 1700 pregnant women in Cambridge showed that a third of all women would not terminate in the event of the detection of a fetal abnormality, a further 7% felt that nobody should, and 10% would rather not know of the presence of an abnormality.

Such screening for good genes is reminiscent of Nazi EUGENICS: Pressure to terminate CF fetuses to reduce the health cost burden is a worry, as a CF patient costs well over £200,000 in care and treatment during his or her lifetime. CF sufferers may feel stigmatised by such a programme and resources could be diverted away from those affected by the disease. False test positives can be disturbing, affecting mother-infant bonding, and screening is regarded by many as playing `Russian roulette' with forbidden knowledge. Cases of apparent non-paternity are found, as up to 15% of the population when tested reveal that the mother's partner may not be the biological father of the child. Finally there is the cost of the screening programme, training and counselling adds to an ever increasing health cost burden to the tax payer.

Perhaps more than other diseases, the study of CF has revolutionized molecular medicine, advancing our understanding of epithelial transport, ion channels and the molecular basis of disease. CF has presented a new generation of ethical and social dilemmas for society to reconcile. In many ways CF is the Down's Syndrome of the 21st Century, for example if we could treat CF patients to make them fertile, should we? If we could, as in common practice in IVF, test all early (4 cell stage embryos) derived from CF carriers for future CF homozygotes, we could feasibly eradicate the disease, its costs and its horrors within a generation. From here it is but a small step to complete disease testing for all conceptions at the embryonic stage, and the potential eradication of all genetically characterized diseases. Why after all, when testing for CF, not perform a general screen. Inherited disease could be wiped from the map, but with them we may eliminate otherwise precious mutations such as delta508 and delta32, ancient protectors against typhoid and bubonic plague epidemics which ravaged Europe. Perhaps inherited disease is the price we pay for that genetic diversity that preserves the human species, ensuring that whatever the future environmental challenge, be it disease or global warming, that we will be around for a good long time yet.

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