79
EDITORIALS
Cystic fibrosis: prospects for screening and therapy Cystic fibrosis (CF) is one of the commonest inherited diseases, affecting approximately 1 in 2500 live births in Caucasian populations. Identification last year of the gene mutated in CF patients1.2 (named cystic fibrosis transmembrane conductance regulator, CFTR) has not only enabled prenatal diagnosis by direct analysis of the mutation3 but also encouraged researchers to contemplate population carrier screening and the possibility of "cure" of patients by somatic gene therapy. The physiology and genetics of the disease have lately been reviewed,4,5 and an earlier editorial (Dec 16, p 1433) discussed the biochemical defect underlying CF. The coding regions of the CFTR gene predict a protein (1480 aminoacids, molecular mass approximately 170 kD) which is probably a member of a large family of eukaryotic membrane-bound transport molecules.2°6These proteins have two hydrophobic domains. The decrease in fluid secretion and accumulation of dehydrated mucus that is the most severe symptom of CF is almost certainly due to altered chloride transport across epithelia (as observed in the diagnostic sweat test). The only mutation in the CFTR gene identified so far is a deletion of three base-pairs resulting in the removal of the phenylalanine residue at position 508 of the mature protein (hence its name F5o8).2,7 This aminoacid is within a prospective ATP-binding domain, and its deletion may prevent nucleotide binding or changes in CFTR conformation that are essential for normal function. Identification of the CFTR gene makes it possible to understand the control of ion transport, and the study of the CFTR protein in its normal and mutant forms should provide a molecular basis for better therapy. A possible method is the creation of a mouse model for CF by homologous recombination of mutant genes into the mouse analogue of CFTR.8 If this can be achieved, it would allow greater understanding of the progress of the disease and provide a safe means of testing new treatments. The ultimate form of treatment for a genetic disease is replacement of the mutated gene by a normal copy-gene therapy.9 This contentious approach has
proposed for haemoglobinopathies,
the treatment of the in which normal genes contained in retroviral vectors are introduced into the bone marrow. The most severely affected organ in CF patients is the lung, and since drugs can easily be administered by aerosol it may be possible to introduce normal CFTR genes, or protein, into the stem cells that differentiate into airway tissue. This approach requires the identification of the cell types within the lung that normally express CFTR and the refinement of methods for introducing DNA to these cells. Now that the CF gene has been pinpointed it is possible to determine its tissue-type and cell-type specific expression.DNA linked to polylysine can be taken in and expressed by eukaryotic cells both in vitro and in vivo. 10 This method obviates the need for retroviral vectors, in which non-specific expression can be difficult to control. If DNA can be introduced regularly via aerosol, there will be no need for integration of the normal gene into host DNA. Exciting prospects may be in the offing. A more immediate way of tackling CF has also been presented by cloning of the gene and identification of the major mutation-population screening. If the unaffected heterozygous carriers of the mutant gene (11 in 25 of the population) can be identified, disease prevention becomes possible by selective termination after prenatal diagnosis in 1 in 4 at-risk pregnancies (when both partners carry the mutant gene). This issue has prompted heated exchanges in correspondence columns lately. 11-13 It is argued that screening for CF heterozygotes should wait until more of the mutations have been identified and the individuals being tested have been sufficiently well educated to understand the results.7,11-13 The frequency of .F 508 on CF chromosomes varies between ethnic groups. In Scotland, this mutation is present on approximately 75 % of CF chromosomes,14 whereas in Spanish and Italian populations it comprises only 49% and 43% of mutations, respectively. 15 If one assumes that the Scottish population is broadly representative of the UK as a whole, 3% of the population would be positive when tested for the deletion. In 3% of these cases their partner will also test positive for the deletion (ie, [1/25 x 3/4]2 =1/1100 couples). Unfortunately, the remainder will have had their risk of having a CF child been
80
increased to 1 in 400 and will therefore require careful counselling and attitude assessment to determine whether this status is acceptable. The effort now being expended worldwide on identification of other mutations should reduce this proportion in the near future. Meanwhile, it seems prudent to instigate pilot studies to determine the best point at which to screen
(eg, antenatal, family planning, or general practice clinic) and to assess how people react to being told their genetic status. Assuming an uptake of 85% among pregnant women (the most likely target group) and 90% among their partners, and detection of 75% of CF mutations, the incidence of CF among those tested could be reduced by over 40%. Since any screening programme will take several years to become fully operational, pilot schemes should be introduced in the very near future. It is important that the mistakes made in the introduction of screening for sickle-cell anaemia are not repeated and that lessons are learnt from the successful screening for p-thalassaemia in Mediterranean populations.16 Detection of heterozygotes carrying the D F 508 mutation is facilitated by the use of polyacrylamide gel electrophoresis, which obviates the need for radioisotopes .’-7,111 Alternatively, the amplification refractory mutation system (ARMS)l9 may prove useful for analysis of this mutation. Thus cloning of the CF gene gives hope for a greater understanding of the disease, for more effective treatment regimens in the long term, and most importantly, for heterozygote screening. Carrier screening should be freely available for two main reasons: (a) it gives prospective parents the chance to make an informed choice, and (b) the reduction in the incidence of CF in the population will free resources for the CF patients alive today. In the UK, screening for the A F508 mutation alone would identify 120 CF fetuses each year out of approximately 700 000 pregnancies in the country as a whole.
11. Stewart AD. Screening for cystic fibrosis. Nature 1989; 341: 696. 12. Ten Kate LP. Carrier screening in CF. Nature 1989; 342: 131. 13. Roderick PJ, Chapple J. Screening for cytstic fibrosis. Lancet 1989; ii: 1403-04. 14. McIntosh I, Lorenzo M-Z, Brock DJH. Frequency of &Dgr;F508 mutation on cystic fibrosis chromosomes in UK. Lancet 1989; ii: 1404-05. 15. Estivill X, Chillon M, Casals T, et al. &Dgr;F508 gene deletion in cystic fibrosis in Southern Europe. Lancet 1989; ii: 1404. 16. The haemoglobinopathies in Europe: combined report on two WHO meetings. Copenhagen: WHO Regional Office for Europe, 1988. 17. Mathew CG, Roberts RG, Harris A, et al. Rapid screening for &Dgr;F508 deletion in cystic fibrosis. Lancet 1989; ii: 1346. 18. Schieffer H, Verlind E, Penninga D, et al. Rapid screening for &Dgr;F508 deletion in cystic fibrosis. Lancet 1989; ii: 1345-46. 19. Newton CR, Heptinstall LE, Summers C, et al. Amplification refractory mutation system for prenatal diagnosis and carrier assessment in cystic fibrosis. Lancet 1989; ii: 1481-83.
in the the numbers
Organ donors
During the past five years services for patients with end-stage renal failure in the UK have improved considerably. By extending the age limits to include the very young and the old, acceptance rates have risen steadilyin 1987, 50 new patients per million of the population (pmp) were taken on for treatment that year.1988 saw a further rise, with some areas, such as Wales, accepting over 70 new patients pmp for treatment.2 Kidney transplant survival rates have also improved to the extent that approximately 80% of first cadaveric kidney transplants can now be expected function for at least a year. Yet there has been no improvement in the supply of cadaveric organs for transplantation. In 1981, Jennett estimated that there were 4000 brainstem deaths annually in the UK,3 and it had been assumed that if sufficient effort was made to identify these individuals as organ donors there would be enough kidneys to supply the dialysis population. However, the annual rate of kidney transplantation only increased from 1443 to 1575 between 1984 and 1988 and transplant activity during 1989 was only marginally better. The waiting list for transplantation continues to increase, and now stands to
at
3684.
The
1. Rommens JM, lanuzzi MC, Kerem B, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989; 245: 1059-65. 2. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-73. 3. McIntosh I, Raeburn JA, Curtis A, Brock DJH. First trimester prenatal diagnosis of cystic fibrosis by direct gene probing. Lancet 1989; ii: 972-73. 4. Boat TF, Welsh MJ, Beaudet AL. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease, 6th ed. New York: McGraw-Hill, 1989: 2649-80. 5. Goodfellow PN, ed. Cystic fibrosis. Oxford: Oxford University Press, 1989. 6. Higgins C. Protein joins transport family. Nature 1989; 341: 103. 7. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1074-80. 8. Cappechi MR. The new mouse genetics: altering the genome by gene targetting. Trends Genet 1989; 5: 70-76. 9. Danielson H. Gene therapy in man. Lancet 1988; i: 1271-72. 10. Wu CH, Wilson JM, Wu GY. Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J Biol Chem 1989; 264: 16985-87.
UK—getting right
1988
represents just 29 kidney which is less than half the rate transplants year pmp, required to match the annual accrual of patients with renal failure. Dr Gore and her colleaguess have lately reported the results of an audit that was carried out in 278 intensive care units in England. Of 2853 deaths recorded during the first quarter of 1989, only 148 patients (5%) became organ donors-ie, 12 donors or
figure
a
24 kidneys pmp. Realisation that the supply of cadaver kidneys might have reached a ceiling has prompted much debate, since, if true, the long-term objectives for the management of patients with renal failure may have to be revised (and recosted). A symposium on organ donation was held in November at the Department of Health, and further discussions took place at a similar symposium in Cardiff, and at the autumn meeting of the British Transplantation Society. At the symposia, some preliminary and as yet unpublished findings were presented from an audit of hospital deaths that had just beeP
EDITORIALS
Cystic fibrosis: prospects for screening and therapy Cystic fibrosis (CF) is one of the commonest inherited diseases, affecting approximately 1 in 2500 live births in Caucasian populations. Identification last year of the gene mutated in CF patients1.2 (named cystic fibrosis transmembrane conductance regulator, CFTR) has not only enabled prenatal diagnosis by direct analysis of the mutation3 but also encouraged researchers to contemplate population carrier screening and the possibility of "cure" of patients by somatic gene therapy. The physiology and genetics of the disease have lately been reviewed,4,5 and an earlier editorial (Dec 16, p 1433) discussed the biochemical defect underlying CF. The coding regions of the CFTR gene predict a protein (1480 aminoacids, molecular mass approximately 170 kD) which is probably a member of a large family of eukaryotic membrane-bound transport molecules.2°6These proteins have two hydrophobic domains. The decrease in fluid secretion and accumulation of dehydrated mucus that is the most severe symptom of CF is almost certainly due to altered chloride transport across epithelia (as observed in the diagnostic sweat test). The only mutation in the CFTR gene identified so far is a deletion of three base-pairs resulting in the removal of the phenylalanine residue at position 508 of the mature protein (hence its name F5o8).2,7 This aminoacid is within a prospective ATP-binding domain, and its deletion may prevent nucleotide binding or changes in CFTR conformation that are essential for normal function. Identification of the CFTR gene makes it possible to understand the control of ion transport, and the study of the CFTR protein in its normal and mutant forms should provide a molecular basis for better therapy. A possible method is the creation of a mouse model for CF by homologous recombination of mutant genes into the mouse analogue of CFTR.8 If this can be achieved, it would allow greater understanding of the progress of the disease and provide a safe means of testing new treatments. The ultimate form of treatment for a genetic disease is replacement of the mutated gene by a normal copy-gene therapy.9 This contentious approach has
proposed for haemoglobinopathies,
the treatment of the in which normal genes contained in retroviral vectors are introduced into the bone marrow. The most severely affected organ in CF patients is the lung, and since drugs can easily be administered by aerosol it may be possible to introduce normal CFTR genes, or protein, into the stem cells that differentiate into airway tissue. This approach requires the identification of the cell types within the lung that normally express CFTR and the refinement of methods for introducing DNA to these cells. Now that the CF gene has been pinpointed it is possible to determine its tissue-type and cell-type specific expression.DNA linked to polylysine can be taken in and expressed by eukaryotic cells both in vitro and in vivo. 10 This method obviates the need for retroviral vectors, in which non-specific expression can be difficult to control. If DNA can be introduced regularly via aerosol, there will be no need for integration of the normal gene into host DNA. Exciting prospects may be in the offing. A more immediate way of tackling CF has also been presented by cloning of the gene and identification of the major mutation-population screening. If the unaffected heterozygous carriers of the mutant gene (11 in 25 of the population) can be identified, disease prevention becomes possible by selective termination after prenatal diagnosis in 1 in 4 at-risk pregnancies (when both partners carry the mutant gene). This issue has prompted heated exchanges in correspondence columns lately. 11-13 It is argued that screening for CF heterozygotes should wait until more of the mutations have been identified and the individuals being tested have been sufficiently well educated to understand the results.7,11-13 The frequency of .F 508 on CF chromosomes varies between ethnic groups. In Scotland, this mutation is present on approximately 75 % of CF chromosomes,14 whereas in Spanish and Italian populations it comprises only 49% and 43% of mutations, respectively. 15 If one assumes that the Scottish population is broadly representative of the UK as a whole, 3% of the population would be positive when tested for the deletion. In 3% of these cases their partner will also test positive for the deletion (ie, [1/25 x 3/4]2 =1/1100 couples). Unfortunately, the remainder will have had their risk of having a CF child been
80
increased to 1 in 400 and will therefore require careful counselling and attitude assessment to determine whether this status is acceptable. The effort now being expended worldwide on identification of other mutations should reduce this proportion in the near future. Meanwhile, it seems prudent to instigate pilot studies to determine the best point at which to screen
(eg, antenatal, family planning, or general practice clinic) and to assess how people react to being told their genetic status. Assuming an uptake of 85% among pregnant women (the most likely target group) and 90% among their partners, and detection of 75% of CF mutations, the incidence of CF among those tested could be reduced by over 40%. Since any screening programme will take several years to become fully operational, pilot schemes should be introduced in the very near future. It is important that the mistakes made in the introduction of screening for sickle-cell anaemia are not repeated and that lessons are learnt from the successful screening for p-thalassaemia in Mediterranean populations.16 Detection of heterozygotes carrying the D F 508 mutation is facilitated by the use of polyacrylamide gel electrophoresis, which obviates the need for radioisotopes .’-7,111 Alternatively, the amplification refractory mutation system (ARMS)l9 may prove useful for analysis of this mutation. Thus cloning of the CF gene gives hope for a greater understanding of the disease, for more effective treatment regimens in the long term, and most importantly, for heterozygote screening. Carrier screening should be freely available for two main reasons: (a) it gives prospective parents the chance to make an informed choice, and (b) the reduction in the incidence of CF in the population will free resources for the CF patients alive today. In the UK, screening for the A F508 mutation alone would identify 120 CF fetuses each year out of approximately 700 000 pregnancies in the country as a whole.
11. Stewart AD. Screening for cystic fibrosis. Nature 1989; 341: 696. 12. Ten Kate LP. Carrier screening in CF. Nature 1989; 342: 131. 13. Roderick PJ, Chapple J. Screening for cytstic fibrosis. Lancet 1989; ii: 1403-04. 14. McIntosh I, Lorenzo M-Z, Brock DJH. Frequency of &Dgr;F508 mutation on cystic fibrosis chromosomes in UK. Lancet 1989; ii: 1404-05. 15. Estivill X, Chillon M, Casals T, et al. &Dgr;F508 gene deletion in cystic fibrosis in Southern Europe. Lancet 1989; ii: 1404. 16. The haemoglobinopathies in Europe: combined report on two WHO meetings. Copenhagen: WHO Regional Office for Europe, 1988. 17. Mathew CG, Roberts RG, Harris A, et al. Rapid screening for &Dgr;F508 deletion in cystic fibrosis. Lancet 1989; ii: 1346. 18. Schieffer H, Verlind E, Penninga D, et al. Rapid screening for &Dgr;F508 deletion in cystic fibrosis. Lancet 1989; ii: 1345-46. 19. Newton CR, Heptinstall LE, Summers C, et al. Amplification refractory mutation system for prenatal diagnosis and carrier assessment in cystic fibrosis. Lancet 1989; ii: 1481-83.
in the the numbers
Organ donors
During the past five years services for patients with end-stage renal failure in the UK have improved considerably. By extending the age limits to include the very young and the old, acceptance rates have risen steadilyin 1987, 50 new patients per million of the population (pmp) were taken on for treatment that year.1988 saw a further rise, with some areas, such as Wales, accepting over 70 new patients pmp for treatment.2 Kidney transplant survival rates have also improved to the extent that approximately 80% of first cadaveric kidney transplants can now be expected function for at least a year. Yet there has been no improvement in the supply of cadaveric organs for transplantation. In 1981, Jennett estimated that there were 4000 brainstem deaths annually in the UK,3 and it had been assumed that if sufficient effort was made to identify these individuals as organ donors there would be enough kidneys to supply the dialysis population. However, the annual rate of kidney transplantation only increased from 1443 to 1575 between 1984 and 1988 and transplant activity during 1989 was only marginally better. The waiting list for transplantation continues to increase, and now stands to
at
3684.
The
1. Rommens JM, lanuzzi MC, Kerem B, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989; 245: 1059-65. 2. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-73. 3. McIntosh I, Raeburn JA, Curtis A, Brock DJH. First trimester prenatal diagnosis of cystic fibrosis by direct gene probing. Lancet 1989; ii: 972-73. 4. Boat TF, Welsh MJ, Beaudet AL. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease, 6th ed. New York: McGraw-Hill, 1989: 2649-80. 5. Goodfellow PN, ed. Cystic fibrosis. Oxford: Oxford University Press, 1989. 6. Higgins C. Protein joins transport family. Nature 1989; 341: 103. 7. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1074-80. 8. Cappechi MR. The new mouse genetics: altering the genome by gene targetting. Trends Genet 1989; 5: 70-76. 9. Danielson H. Gene therapy in man. Lancet 1988; i: 1271-72. 10. Wu CH, Wilson JM, Wu GY. Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J Biol Chem 1989; 264: 16985-87.
UK—getting right
1988
represents just 29 kidney which is less than half the rate transplants year pmp, required to match the annual accrual of patients with renal failure. Dr Gore and her colleaguess have lately reported the results of an audit that was carried out in 278 intensive care units in England. Of 2853 deaths recorded during the first quarter of 1989, only 148 patients (5%) became organ donors-ie, 12 donors or
figure
a
24 kidneys pmp. Realisation that the supply of cadaver kidneys might have reached a ceiling has prompted much debate, since, if true, the long-term objectives for the management of patients with renal failure may have to be revised (and recosted). A symposium on organ donation was held in November at the Department of Health, and further discussions took place at a similar symposium in Cardiff, and at the autumn meeting of the British Transplantation Society. At the symposia, some preliminary and as yet unpublished findings were presented from an audit of hospital deaths that had just beeP
