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Genetics of cystic fibrosis
Mini-symposium
Respiratory
Genetics of cystic fibrosis
J. Hull, A. H. Thomson Cystic fibrosis (CF) is a complex, inherited disorder affecting children, many of whom now live into adulthood. It remains the most common life threatening recessive genetic disorder in the Caucasian population and there are over 6000 people with CF in the UK. It is a disease primarily of epithelial tissues and affects the airway epithelium, the pancreatic duct, genital tract, the biliary tree and the sweat glands. Most of the morbidity and mortality in CF results from damage to the lung and gastro-intestinal tract. There is a wide range of disease severity but all affected people need daily treatment and in the majority CF has a marked influence on their lifestyle. Without treatment the mean life expectancy is 3-5 years. With current therapy and optimal treatment, infants born today with CF are thought to have a mean life expectancy of 35-40 years. It is hoped that increased understanding of the molecular basis of CF will improve both the quality, and the length, of the lives of affected individuals.
+-
Plasma membrane
phosphotylation site
Fig. 1-Model of the predicted structure of the CFTR protein. MSD=membrane spanning domain, NBF=nucleotide binding fold, R = regulatory domain.
CFTR function
CF is an autosomal recessive disorder. The gene that is defective in CF was identified in 1989l~~*~and has been called the cystic Jibrosis transmembrane conductance regulator (CFTR) gene. It is located on chromosome 7 (7q3 1) and is a large gene, spanning over 230kb of genomic DNA and containing 27 exons. The predicted structure of the protein product, based on the primary amino acid sequence is shown in Figure 1. This structure resembles that of a family of proteins known to function as active transporters of molecules across cell membranes. Despite this resemblance, CFTR has not been demonstrated to have such a function, and in fact there is now compelling evidence that CFTR is a small chloride channel located at the apical surface of several epithelia.
The CFTR chloride channel opens in response to CAMP resulting in chloride secretion. The secreted chloride draws water, by osmosis, across the epithelium and into the lumen. In CF tissues this CAMP stimulated chloride secretion is absent. Currently, the most popular model for the pathogenesis of CF suggests that the diminished chloride and water secretion of CF epithelial tissues results in the formation of dehydrated and therefore viscous mucus. In the lungs, the thicker mucus prevents normal ciliary function and collects in the airways, eventually becoming infected. The inflammation resulting from the infection, as well as the infecting agents themselves, cause progressive damage to the airway. In the pancreas, the thick mucus obstructs the pancreatic ducts thus preventing secretion of pancreatic enzymes. The enzymes escape the normal inhibition of their activity and destroy the pancreas.
Jeremy Hull MRCP, Research Fellow, Anne Thomson MD, MRCP,
Current Poediarrics (1994) 4, 136-138 0 1994 Longman Group Ltd
MSD 2
Cytoplasm
Molecular genetics
Consultant Paediatrician, Department of Paediatrics, Radcliffe Hospital, Headington, Oxford OX3 9DU. Correspondence and requests for offprints to JH.
polysaccharide
Outside cell
John
136
GENETICS OF CYSTIC FIBROSIS
Whether or not this model is a sufficient explanation of all the abnormalities in CF remains open to debate. Nevertheless it has provided the impetus for a number of novel therapies in CF to try and increase the chloride and hence fluid secretion of these epithelia. It is worth mentioning that in addition to defective chloride secretion, the CF airway uniquely shows excessive sodium absorption. Quantitatively this is a much more significant possible cause of airway mucus dehydration. There is no convincing mechanism which can explain the cause of the excess sodium absorption based on the known functions of CFTR. The discovery of the excessive sodium absorption led to studies into the possible beneficial effects of sodium channel blockers, particularly inhaled amiloride.
Mutations in the CFTR gene In the UK, one mutation, a 3 base pair deletion called AF508, is found on approximately 80% of CF chromosomes. This means that about 50-60% of UK CF patients are homozygous for this mutation. A further 25% have AF508 on one chromosome and a different mutation on the other chromosome, and the remainder have non- F508 mutations on both chromosomes. To date over 350 different mutations in CFTR have been reported to the Cystic Fibrosis Genetics Analysis Consortium. The majority of these are present in only one or a few patients. Table 1 lists 5 of the most common mutations found in the UK. The existence of a large number of different CFTR mutations makes foolproof population screening for CF impossible (see below). The frequency of CFTR mutations varies in different parts of the world.
Genotype-phenotype correlations There is a wide range of disease severity in individuals with CF. After the identification of the CFTR gene and some of the common mutations, there was much interest to try and establish whether particular mutations (genotype) were associated with differing degrees of severity of the disease (phenotype). It was hoped that this would not only increase understanding of the normal function of CFTR, but would also allow clinicians to predict the likely course of the disease in very young children. Unfortunately the results of these studies have been disappointing. Although a number of mutations have been identified that do seem to predict pancreatic status,4 for other phenotypic differences, such as pulmonary function, likelihood of Pseudomonas colonisation, age at Table 1 Frequency the UK population
of each mutation
of CF chromosomes
in
137
diagnosis, sweat chloride levels and nutritional status, the associations are less clear cut, and some of the differences that have been reported may simply reflect the pancreatic status of the patient.’ The earlier observation that patients homozygous for nonsense mutations had mild forms of the disease6 does not seem to be universal.
Diagnosis Despite the advances and knowledge in the molecular genetics of CF, the diagnosis remains largely clinical. Children who show persistent weight loss or who have recurrent coughs or evidence of repeated pulmonary infections should have sweat tests. There have been very few reported cases where sweat chloride has been normal in the face of other convincing evidence of CF, such as an abnormal nasal potential difference (CF patients have a more negative PD across their nasal epithelium), and the presence of two CF mutations. In other words a sweat test, conducted well, remains the basis for diagnosis. Sending blood for mutation analysis is of limited value, since it is practical to screen for only a few of the common mutations (Table 1) and so currently genetic analysis will only pick up approximately 80% of individuals with CF. A positive result is therefore useful but a negative result or the identification of one CF mutation does not help make the diagnosis in equivocal cases.
Carrier screening The high carrier frequency of the CF gene in the British population has led to trials of carrier screening despite the knowledge that approximately 15% of carriers will give a negative result on simple mutation screening. In the UK one partner is a carrier in every 12 couples and in one in 540 couples both partners are carriers and they therefore have a one in four risk in each pregnancy of producing an affected child. It is theoretically possible to identify and offer prenatal diagnosis in approximately 75% of these carrier couples. The Cystic Fibrosis Research Trust has funded a number of projects to assess the acceptability and uptake of population screening in the UK. Bekker reported on the uptake of CF carrier testing in primary care.’ Screening was offered to people of reproductive age but uptake when invited by letter or given information about CF was very poor (4-l 7%) and no carrier couples were identified after almost 1000 tests, indicating that population screening was inefficient. Other studies have concentrated on screening during early pregnancy and achieve uptake rates of 84-90”h.8’9 Studies in Edinburgh have taken samples from both partners at one visit. The mother’s sample was tested first and, if positive, the partner’s sample was tested before conveying the result to the couple. This approach avoided the anxiety produced when the mother knew she was positive and was waiting
138 CURRENT PAEDIATRICS for her partner’s test result. If both partners were positive, they were offered chorionic villus sampling to test the fetus. Any screening programme requires adequate counselling both before and after testing. In the context of CF where the test fails to identify 15% of carriers, counselling is time consuming. A nationwide screening programme could significantly reduce the number of CF births if the decision to terminate pregnancies were taken. However, with new therapies likely to be effective in the treatment of CF and potential life expectancy of 40 years, the ethics of terminating affected fetuses need to be debated widely.
Neonatal screening Neonatal screening for CF is by no means universal in the UK. There is as yet no convincing data that children diagnosed as neonates have a better longterm outcome. There are studies however suggesting that diagnosis by screening and early treatment improve short-term morbidity.“*” In addition there are those who would argue the benefits to the family of early diagnosis, which includes the possibility of screening future pregnancies, is sufficient to justify neonatal screening. A number of methods of screening have been attempted. The most successful employ measurement of immuno-reactive trypsinogen (IRT). This can be measured from an additional blood spot on the Guthrie testing card. High levels of trypsinogen, probably due to leakage from the obstructed pancreas, are found in the blood of neonates with CF. IRT levels then fall to normal a few weeks or months after birth. The false negative rate (other than for infants with meconium ileus) for the IRT test is very low. The false positive rate is significant (approximately 0.5%), and many screening centres adopt a two tier system of retesting positive infants within 6-8 weeks. Since the IRT falls with age this is not always successful and may lead to failure to diagnose some infants with CF. An alternative to repeat IRT testing is to perform genetic analysis for the common mutations on the same spot of blood used for IRT analysis. If a mutation is detected (in single or double copy) the patient is called back for a sweat test. This combined approach significantly reduces the false positive rate and the number of sweat tests needed. By reducing the call back rate it also keeps parental anxiety to a minimum. In one study the call back rate was only 2.8 families for every child diagnosed.” Only a very small proportion of CF children were missed by this screening method.
Gene therapy There are many studies investigating novel therapies for CF. The most exciting, and potentially the most effective of these is the possibility of introducing normal copies of the CFTR gene into epithelial cells.
Very recently trials of gene therapy have been started in the USA and the UK. Both are aimed at introducing normal copies of the CFTR gene into the airway of patients with CF. This form of gene therapy is called somatic gene therapy and is not designed to introduce the gene into the germ line. Any genetic changes cannot then be passed on to future generations. One of the difficulties of gene therapy in the airway is that no stem cell can be identified. This means that when the treated cells die and detach from the epithelium, the cells that grow up to replace them do not carry a normal copy of the CFTR gene. Any gene therapy which uses this approach will therefore have to be repeated, possibly as often as every 2-4 weeks. Gene therapy is then simply another treatment, and not a cure. The best method of introduction of a normal gene into the airway is being sought in these early trials. In the USA adenoviruses are being used; the UK trials are using a liposome delivery system. It is important to remember that these are phase 1 trials. Much larger and longer trials will be needed to establish whether this form of therapy will be effective in halting lung destruction in patients with CF. There has been an enormous advance in our understanding of the genetics of CF over the past 5 years. The challenge for the next decade is to translate this into progress in practical management of the disease.
References 1. Kerem B, Rommens JM, Buchanan JA et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1083-1080. 2. Riordan JR, Rommens JM, Kerem BS et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-1073. 3. Rommens JM, Iannuzzi MC, Kerem B et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989; 245: 1059-1065. 4. Kristidis P, Bozon D, Corey M et al. Genetic determination of exocrine pancreatic function in cystic fibrosis. Am J Hum Genet 1992; 50: 1178-l 184. 5. Kubesch P, Dork T, Wulbrand U et al. Genetic determinants of airways colonisation with Pseudomonas aeruginosa in cystic fibrosis. Lancet 1993; 341: 189-193. 6. Cutting GR, Kasch LM, Rosenstein BJ, Tsui L-C, Kazazian HH, Antonarakis SE. Two patients with cystic fibrosis, nonsense mutations in each cystic fibrosis gene, and mild pulmonary disease. New Eng J Med 1990; 323: 168551689. 7. Bekker H, Model1 M, Denniss G et al. Uptake of cystic fibrosis carriers testing in primary care: supply push or demand pull? BMJ 1993; 306: 1584-1587. 8. Mennie ME, Gilfillan A, Compton M et al. Prenatal screening for cystic fibrosis. Lancet 1992; 340: 214-216. 9. Harris H, Scotcher D, Hartley N, Wallace A, Craufard D, Harris R. Cystic fibrosis carrier testing in early pregnancy by general practitioners, BMJ 1993; 306: 1580-1583. 10. Wilcken B, Chalmers G. Reduced morbidity in patients with cystic fibrosis detailed by neonatal screening. Lancet 1985; ii: 1319-1321. 11, Weaver LT, Green MR, Nicholson K et al. Prognosis in cystic fibrosis treated with continuous flucloxacillin from the neonatal period. Arch Dis Child 1994; 70: 84-89. 12. Ranier E, Ryall RG, Morris CP et al. Neonatal screening strategy for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis BMJ 1991; 302: 1237-1240.
Respiratory
Genetics of cystic fibrosis
J. Hull, A. H. Thomson Cystic fibrosis (CF) is a complex, inherited disorder affecting children, many of whom now live into adulthood. It remains the most common life threatening recessive genetic disorder in the Caucasian population and there are over 6000 people with CF in the UK. It is a disease primarily of epithelial tissues and affects the airway epithelium, the pancreatic duct, genital tract, the biliary tree and the sweat glands. Most of the morbidity and mortality in CF results from damage to the lung and gastro-intestinal tract. There is a wide range of disease severity but all affected people need daily treatment and in the majority CF has a marked influence on their lifestyle. Without treatment the mean life expectancy is 3-5 years. With current therapy and optimal treatment, infants born today with CF are thought to have a mean life expectancy of 35-40 years. It is hoped that increased understanding of the molecular basis of CF will improve both the quality, and the length, of the lives of affected individuals.
+-
Plasma membrane
phosphotylation site
Fig. 1-Model of the predicted structure of the CFTR protein. MSD=membrane spanning domain, NBF=nucleotide binding fold, R = regulatory domain.
CFTR function
CF is an autosomal recessive disorder. The gene that is defective in CF was identified in 1989l~~*~and has been called the cystic Jibrosis transmembrane conductance regulator (CFTR) gene. It is located on chromosome 7 (7q3 1) and is a large gene, spanning over 230kb of genomic DNA and containing 27 exons. The predicted structure of the protein product, based on the primary amino acid sequence is shown in Figure 1. This structure resembles that of a family of proteins known to function as active transporters of molecules across cell membranes. Despite this resemblance, CFTR has not been demonstrated to have such a function, and in fact there is now compelling evidence that CFTR is a small chloride channel located at the apical surface of several epithelia.
The CFTR chloride channel opens in response to CAMP resulting in chloride secretion. The secreted chloride draws water, by osmosis, across the epithelium and into the lumen. In CF tissues this CAMP stimulated chloride secretion is absent. Currently, the most popular model for the pathogenesis of CF suggests that the diminished chloride and water secretion of CF epithelial tissues results in the formation of dehydrated and therefore viscous mucus. In the lungs, the thicker mucus prevents normal ciliary function and collects in the airways, eventually becoming infected. The inflammation resulting from the infection, as well as the infecting agents themselves, cause progressive damage to the airway. In the pancreas, the thick mucus obstructs the pancreatic ducts thus preventing secretion of pancreatic enzymes. The enzymes escape the normal inhibition of their activity and destroy the pancreas.
Jeremy Hull MRCP, Research Fellow, Anne Thomson MD, MRCP,
Current Poediarrics (1994) 4, 136-138 0 1994 Longman Group Ltd
MSD 2
Cytoplasm
Molecular genetics
Consultant Paediatrician, Department of Paediatrics, Radcliffe Hospital, Headington, Oxford OX3 9DU. Correspondence and requests for offprints to JH.
polysaccharide
Outside cell
John
136
GENETICS OF CYSTIC FIBROSIS
Whether or not this model is a sufficient explanation of all the abnormalities in CF remains open to debate. Nevertheless it has provided the impetus for a number of novel therapies in CF to try and increase the chloride and hence fluid secretion of these epithelia. It is worth mentioning that in addition to defective chloride secretion, the CF airway uniquely shows excessive sodium absorption. Quantitatively this is a much more significant possible cause of airway mucus dehydration. There is no convincing mechanism which can explain the cause of the excess sodium absorption based on the known functions of CFTR. The discovery of the excessive sodium absorption led to studies into the possible beneficial effects of sodium channel blockers, particularly inhaled amiloride.
Mutations in the CFTR gene In the UK, one mutation, a 3 base pair deletion called AF508, is found on approximately 80% of CF chromosomes. This means that about 50-60% of UK CF patients are homozygous for this mutation. A further 25% have AF508 on one chromosome and a different mutation on the other chromosome, and the remainder have non- F508 mutations on both chromosomes. To date over 350 different mutations in CFTR have been reported to the Cystic Fibrosis Genetics Analysis Consortium. The majority of these are present in only one or a few patients. Table 1 lists 5 of the most common mutations found in the UK. The existence of a large number of different CFTR mutations makes foolproof population screening for CF impossible (see below). The frequency of CFTR mutations varies in different parts of the world.
Genotype-phenotype correlations There is a wide range of disease severity in individuals with CF. After the identification of the CFTR gene and some of the common mutations, there was much interest to try and establish whether particular mutations (genotype) were associated with differing degrees of severity of the disease (phenotype). It was hoped that this would not only increase understanding of the normal function of CFTR, but would also allow clinicians to predict the likely course of the disease in very young children. Unfortunately the results of these studies have been disappointing. Although a number of mutations have been identified that do seem to predict pancreatic status,4 for other phenotypic differences, such as pulmonary function, likelihood of Pseudomonas colonisation, age at Table 1 Frequency the UK population
of each mutation
of CF chromosomes
in
137
diagnosis, sweat chloride levels and nutritional status, the associations are less clear cut, and some of the differences that have been reported may simply reflect the pancreatic status of the patient.’ The earlier observation that patients homozygous for nonsense mutations had mild forms of the disease6 does not seem to be universal.
Diagnosis Despite the advances and knowledge in the molecular genetics of CF, the diagnosis remains largely clinical. Children who show persistent weight loss or who have recurrent coughs or evidence of repeated pulmonary infections should have sweat tests. There have been very few reported cases where sweat chloride has been normal in the face of other convincing evidence of CF, such as an abnormal nasal potential difference (CF patients have a more negative PD across their nasal epithelium), and the presence of two CF mutations. In other words a sweat test, conducted well, remains the basis for diagnosis. Sending blood for mutation analysis is of limited value, since it is practical to screen for only a few of the common mutations (Table 1) and so currently genetic analysis will only pick up approximately 80% of individuals with CF. A positive result is therefore useful but a negative result or the identification of one CF mutation does not help make the diagnosis in equivocal cases.
Carrier screening The high carrier frequency of the CF gene in the British population has led to trials of carrier screening despite the knowledge that approximately 15% of carriers will give a negative result on simple mutation screening. In the UK one partner is a carrier in every 12 couples and in one in 540 couples both partners are carriers and they therefore have a one in four risk in each pregnancy of producing an affected child. It is theoretically possible to identify and offer prenatal diagnosis in approximately 75% of these carrier couples. The Cystic Fibrosis Research Trust has funded a number of projects to assess the acceptability and uptake of population screening in the UK. Bekker reported on the uptake of CF carrier testing in primary care.’ Screening was offered to people of reproductive age but uptake when invited by letter or given information about CF was very poor (4-l 7%) and no carrier couples were identified after almost 1000 tests, indicating that population screening was inefficient. Other studies have concentrated on screening during early pregnancy and achieve uptake rates of 84-90”h.8’9 Studies in Edinburgh have taken samples from both partners at one visit. The mother’s sample was tested first and, if positive, the partner’s sample was tested before conveying the result to the couple. This approach avoided the anxiety produced when the mother knew she was positive and was waiting
138 CURRENT PAEDIATRICS for her partner’s test result. If both partners were positive, they were offered chorionic villus sampling to test the fetus. Any screening programme requires adequate counselling both before and after testing. In the context of CF where the test fails to identify 15% of carriers, counselling is time consuming. A nationwide screening programme could significantly reduce the number of CF births if the decision to terminate pregnancies were taken. However, with new therapies likely to be effective in the treatment of CF and potential life expectancy of 40 years, the ethics of terminating affected fetuses need to be debated widely.
Neonatal screening Neonatal screening for CF is by no means universal in the UK. There is as yet no convincing data that children diagnosed as neonates have a better longterm outcome. There are studies however suggesting that diagnosis by screening and early treatment improve short-term morbidity.“*” In addition there are those who would argue the benefits to the family of early diagnosis, which includes the possibility of screening future pregnancies, is sufficient to justify neonatal screening. A number of methods of screening have been attempted. The most successful employ measurement of immuno-reactive trypsinogen (IRT). This can be measured from an additional blood spot on the Guthrie testing card. High levels of trypsinogen, probably due to leakage from the obstructed pancreas, are found in the blood of neonates with CF. IRT levels then fall to normal a few weeks or months after birth. The false negative rate (other than for infants with meconium ileus) for the IRT test is very low. The false positive rate is significant (approximately 0.5%), and many screening centres adopt a two tier system of retesting positive infants within 6-8 weeks. Since the IRT falls with age this is not always successful and may lead to failure to diagnose some infants with CF. An alternative to repeat IRT testing is to perform genetic analysis for the common mutations on the same spot of blood used for IRT analysis. If a mutation is detected (in single or double copy) the patient is called back for a sweat test. This combined approach significantly reduces the false positive rate and the number of sweat tests needed. By reducing the call back rate it also keeps parental anxiety to a minimum. In one study the call back rate was only 2.8 families for every child diagnosed.” Only a very small proportion of CF children were missed by this screening method.
Gene therapy There are many studies investigating novel therapies for CF. The most exciting, and potentially the most effective of these is the possibility of introducing normal copies of the CFTR gene into epithelial cells.
Very recently trials of gene therapy have been started in the USA and the UK. Both are aimed at introducing normal copies of the CFTR gene into the airway of patients with CF. This form of gene therapy is called somatic gene therapy and is not designed to introduce the gene into the germ line. Any genetic changes cannot then be passed on to future generations. One of the difficulties of gene therapy in the airway is that no stem cell can be identified. This means that when the treated cells die and detach from the epithelium, the cells that grow up to replace them do not carry a normal copy of the CFTR gene. Any gene therapy which uses this approach will therefore have to be repeated, possibly as often as every 2-4 weeks. Gene therapy is then simply another treatment, and not a cure. The best method of introduction of a normal gene into the airway is being sought in these early trials. In the USA adenoviruses are being used; the UK trials are using a liposome delivery system. It is important to remember that these are phase 1 trials. Much larger and longer trials will be needed to establish whether this form of therapy will be effective in halting lung destruction in patients with CF. There has been an enormous advance in our understanding of the genetics of CF over the past 5 years. The challenge for the next decade is to translate this into progress in practical management of the disease.
References 1. Kerem B, Rommens JM, Buchanan JA et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1083-1080. 2. Riordan JR, Rommens JM, Kerem BS et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-1073. 3. Rommens JM, Iannuzzi MC, Kerem B et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989; 245: 1059-1065. 4. Kristidis P, Bozon D, Corey M et al. Genetic determination of exocrine pancreatic function in cystic fibrosis. Am J Hum Genet 1992; 50: 1178-l 184. 5. Kubesch P, Dork T, Wulbrand U et al. Genetic determinants of airways colonisation with Pseudomonas aeruginosa in cystic fibrosis. Lancet 1993; 341: 189-193. 6. Cutting GR, Kasch LM, Rosenstein BJ, Tsui L-C, Kazazian HH, Antonarakis SE. Two patients with cystic fibrosis, nonsense mutations in each cystic fibrosis gene, and mild pulmonary disease. New Eng J Med 1990; 323: 168551689. 7. Bekker H, Model1 M, Denniss G et al. Uptake of cystic fibrosis carriers testing in primary care: supply push or demand pull? BMJ 1993; 306: 1584-1587. 8. Mennie ME, Gilfillan A, Compton M et al. Prenatal screening for cystic fibrosis. Lancet 1992; 340: 214-216. 9. Harris H, Scotcher D, Hartley N, Wallace A, Craufard D, Harris R. Cystic fibrosis carrier testing in early pregnancy by general practitioners, BMJ 1993; 306: 1580-1583. 10. Wilcken B, Chalmers G. Reduced morbidity in patients with cystic fibrosis detailed by neonatal screening. Lancet 1985; ii: 1319-1321. 11, Weaver LT, Green MR, Nicholson K et al. Prognosis in cystic fibrosis treated with continuous flucloxacillin from the neonatal period. Arch Dis Child 1994; 70: 84-89. 12. Ranier E, Ryall RG, Morris CP et al. Neonatal screening strategy for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis BMJ 1991; 302: 1237-1240.