- Email: [email protected]
Couple screening for cystic fibrosis
1318
Layward L, Levinsky RJ, Butler M. Long term abnormalities in T and B lymphocyte function in children following treatment for acute lymphoblastic leukemia. Br J Haematol 1981; 49: 251-58. 25. Jermy A, Lilleyman JS, Jennings R, Rees RC. Spontaneous natural killer cell activity in childhood acute lymphoblastic leukaemia. Eur J Cancer 24.
Clin Oncol 1987; 23: 1365-70. MS, Ridgway D. Prolonged defects of interleukin-2 production responsiveness and receptor expression in patients with acute lymphoblastic leukemia. Blood 1989; 73: 1608-14. 27. Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Excerpta Medica, 1982. 26. Borzy
28. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantations. Blood 1990; 75: 555-62. 29. Osmond DG. B cell development in bone marrow. Semin Immunol 1990; 2: 173-80. 30. Gallagher RB, Osmond DG. To B or not to B: that is the question. Immunol Today 1991; 12: 1-3.
31.
Tonegawa S. Somatic generation of antibody diversity. Nature 1983; 302:
575-81. 32. Patte C, Philip T, Rodary C, et al. Improved survival rate in children with stage IV B cell non-Hodgkin lymphoma and leukemia using multiagent chemotherapy: results of a study of 114 children from French Pediatric Oncology Society. J Clin Oncol 1986; 4: 1219-26. 33. Hann M, Eden OB, Barnes J, Pinkerton CR, on behalf of the United Kingdom Children’s Cancer Study Group (UKCCSG). MACHO chemotherapy for stage VI B cell lymphoma and B cell acute lymphoblastic leukemia of childhood. Br J Haematol 1990; 76: 359-64. 34. Barlogie B, Epstein H, Salvanayagam P, Alexanian R. Plasma cell myeloma: new biological insight and advances in therapy. Blood 1989; 73: 865-79. 35. Beisehuizen A, Hahlen K, van Wering ER, et al. Detection of minimal residual disease in childhood leukemia with the polymerase chain reaction. N Engl J Med 1991; 342: 772-74. 36. Greaves MF. Speculation on the cause of childhood acute lymphoblastic leukemia. Leukemia 1988; 2: 120-25.
VIEWPOINT
Couple screening for cystic fibrosis The main aim of screening for congenital disorders is the prevention of handicap with the least harm from side-effects (anxiety and unnecessary invasive procedures) and at the lowest cost for a given level of prevention. It is best achieved by delaying screening until the latest time when effective preventive action can be taken and by choosing an occasion when all people to be screened are likely to be accessible-at what might be termed a natural "screening turnstile", such as birth, school entry, or in early pregnancy. The outlook for patients with cystic fibrosis has been improved by the treatment of complications, but their expectation of life is still less than 25 years. There is substantial associated physical handicap’ and no known cure. An appropriate screening turnstile for cystic fibrosis is, therefore, early pregnancy. The expectant parents have the choice of antenatal screening and, if an affected fetus is identified, the option to terminate the pregnancy. Pregnancy is not only an appropriate turnstile but also a critical one, in that the result will be of immediate importance to the
expectant parents. With autosomal recessive disorders such as cystic fibrosis, only the homozygote with two allelic mutations is affected; the heterozygote with one is unaffected. Since an affected fetus can arise only when both partners each contribute a cystic fibrosis mutation, the screening unit is the couple, not the individual parent. Heterozygotes can be identified easily-all that is needed is a mouthwash sample of buccal cells for DNA analysis collected in a small tube that can be posted to the laboratory by the subject. In screening, the analysis need only be carried out if samples are received from both partners, because both are needed to determine whether a couple is at risk of having an affected fetus. However, in almost all cases only one sample (either will do) is tested, because there is no need to test the second if the first is negative. If both samples have one of the cystic fibrosis mutations tested for, the couple is designated "screen positive" and notified of a positive result; otherwise, the couple is designated "screen negative", with an option of receiving a negative report if they provide a pre-addressed envelope. All couples are, of course, informed of the procedure and the fact that screening is directed at both partners together.
Fig 1 shows the results of couple screening for cystic fibrosis and fig 2 the alternative stepwise approach, in which the mother is screened first and, if she is found to be heterozygous, the father is approached for a sample. The examples describe 250 000 pregnancies with a birth prevalence of 1 in 2500, which implies a heterozygote prevalence of 4% (4% x 4% x 0-25 = 1/2500). It is also assumed that 80% of all heterozygotes, and therefore 64% of all homozygotes (80% x 80%), can be identified. This identification rate is realistic; although more than 150 cystic fibrosis mutations have been identified, 1 (AF508) accounts for about 75% of alleles in Britain2 and another (G551D) accounts for a further 5%.3 In figs 1 and 2, a positive test is one in which a cystic fibrosis mutation is detected. The detection rate (sensitivity) is the proportion of all affected pregnancies that are positive (ie, both parents are found to be heterozygotes) and the false-positive rate is the proportion of all unaffected pregnancies that are
positive. There
important advantages of couple screening stepwise screening. (a) For the same detection rate (64%), the number of women who would be made anxious by the knowledge that they were screen positive is reduced by more than 97% (initial false-positive rate 0-08% vs 3-2%). (b) The odds that a positive initial result is a true positive increase 33-fold (64/192 or 1/3 vs 80/7920 or 1/99). (c) A definitive diagnostic test is available for all screen-positive couples (identification, in the fetus, of the same cystic fibrosis mutations as those found in the parents) which will, with the exception of rare technical errors, yield no false-positive or false-negative results. There is no definitive diagnostic test for all screen-positive women in stepwise screening. In stepwise screening, nearly every woman who is positive will have a negative partner. But even when he is shown to be negative, the position remains uncertain. The are
several
over
woman cannot
be reassured, since she is left with the
ADDRESS Department of Environmental and Preventive Medicine, Wolfson Institute of Preventive Medicine, Medical College of St Bartholomew’s Hospital, Charterhouse Square, London EC1 M 6BQ, UK (Prof N J Wald, FRCP)
1319
Fig 1-Couple screening for cystic fibrosis.
knowledge that because she is heterozygous, there is a
1 in 500 chance that the fetus is affected (80-64/7920-192); the father may have one of the mutations that was not included in the screening test. This is a low risk, but it is five times higher than the general risk of 1 in 2500. The antenatal diagnosis of cystic fibrosis in such pregnancies is difficult and subject to error. If the fetus did not have the mutation identified in the mother, it would be unaffected; but if the maternal mutation were present in the fetus, the chance that the fetus would have cystic fibrosis is about 1 in 250 when the father is screen negative. The use of the microvillar enzyme test4 on amniotic fluid would improve the discrimination. The test can detect 95% of all affected fetuses with a cutoff level that would also identify 8% of unaffected pregnancies as positive. The odds of having an affected fetus would therefore be 1 in 20, so that although the detection rate could be increased from 64% to nearly 80% (fig 2), the cost would be termination of 20 unaffected pregnancies for each cystic fibrosis pregnancy. If the microvillar enzyme test were negative, the odds would be only 1 in 4400. An advantage of couple screening is that it avoids the unnecessary worry that can arise if a woman is found to be a heterozygote but her partner is not available.
A minor disadvantage of couple screening is that samples for DNA analysis are required from both partners (even though the second of each pair is tested only when the test reveals a cystic fibrosis mutation in the first), whereas with stepwise screening they are required only from the women and from about 3-4% of men. Since the specimens needed are aqueous suspensions of buccal cells, the extra cost is negligible; the practice of obtaining samples from both partners before testing ensures that both members of the couple agree to be screened at the outset. Although the uptake may be low because couples might not take the trouble to post their samples to the laboratory, this has not been our experience. A random sample of women who inquired about serum screening for Down’s syndrome were informed about cystic fibrosis couple screening and were sent details together with sample tubes and a reply-paid package; the uptake was 75% (70/94). None of the couples screened asked to know whether the individual tested was a carrier, so inquiries about whether one or other partner is a carrier are likely to be rare. In the event that one partner may wish to know his or her carrier status, he or she would be asked to attend to discuss the request and, if necessary, to be tested. That individual might not have been the one who was tested previously. Antenatal couple screening is therefore a practical and efficient method of screening for cystic fibrosis, but it needs to be implemented on a wider scale and among other, perhaps less motivated, groups of antenatal patients before being introduced into routine obstetric care. The two existing antenatal screening procedures for serious congenital disorders are alphafetoprotein (AFP) screening for open spina bifida, and serum screening for Down’s syndrome by means of AFP, unconjugated oestriol, and human chorionic gonadotropin measurement interpreted in conjunction with maternal age. AFP screening for spina bifida has a detection rate of about 75% with a false-positive rate of 2-3%.s Serum screening for Down’s syndrome has a detection rate of 60% with a false-positive rate of 5%.6 Antenatal couple screening for cystic fibrosis has a detection rate of about 64% and a false-positive rate of 01%, so it is the most effective of the three screening programmes, achieving comparable detection rates at less than a tenth of the false-positive rate. I thank Professor Howard Cuckle and Professor David Brock for useful discussions during the development of the couple screening concept; Dr Ephraim Gazit, Professor Bob Williamson, Dr Caroline Williams, and Ms Annette Gilfilliln for technical help in introducing the DNA technology into our laboratory; Ms Lynne George for the DNA analyses; Ms Karen Fordham and Ms Anne Kennard with the other members of the antenatal screening staff in the department for organising the implementation of the screening service; and Professor Eva Alberman for commenting on the paper.
REFERENCES
Fig 2-Stepwise screening for cystic fibrosis.
1. The British Paediatric Association Working Party on Cystic Fibrosis. Cystic fibrosis in the United Kingdom 1977-85: an improving picture. Br Med J 1988; 297; 1599-602. 2. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1073-80. 3. Cutting CR, Kasch LM, Rosenstein BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide binding fold of the cystic fibrosis conductance regulator protein. Nature 1990; 346: 366-69. 4. Brock DJH, Mennie ME, McIntosh I, Jones C, Shrimpton AE. Heterozygote screening for cystic fibrosis. In: Drife JO, Donnai D, eds. Antenatal diagnosis of fetal abnormalities. London: Springer-Verlag, 1991: 59-70. 5. Wald NJ, Cuckle HS. Biochemical detection of neural tube defects and Down’s syndrome. In: Turnbull A, Chamberlain G, eds. Obstetrics. Edinburgh: Churchill Livingstone, 1989: 269-89. 6. Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum screening for Down’s syndrome in early pregnancy. Br Med J 1988; 297: 883-87.
Layward L, Levinsky RJ, Butler M. Long term abnormalities in T and B lymphocyte function in children following treatment for acute lymphoblastic leukemia. Br J Haematol 1981; 49: 251-58. 25. Jermy A, Lilleyman JS, Jennings R, Rees RC. Spontaneous natural killer cell activity in childhood acute lymphoblastic leukaemia. Eur J Cancer 24.
Clin Oncol 1987; 23: 1365-70. MS, Ridgway D. Prolonged defects of interleukin-2 production responsiveness and receptor expression in patients with acute lymphoblastic leukemia. Blood 1989; 73: 1608-14. 27. Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Excerpta Medica, 1982. 26. Borzy
28. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantations. Blood 1990; 75: 555-62. 29. Osmond DG. B cell development in bone marrow. Semin Immunol 1990; 2: 173-80. 30. Gallagher RB, Osmond DG. To B or not to B: that is the question. Immunol Today 1991; 12: 1-3.
31.
Tonegawa S. Somatic generation of antibody diversity. Nature 1983; 302:
575-81. 32. Patte C, Philip T, Rodary C, et al. Improved survival rate in children with stage IV B cell non-Hodgkin lymphoma and leukemia using multiagent chemotherapy: results of a study of 114 children from French Pediatric Oncology Society. J Clin Oncol 1986; 4: 1219-26. 33. Hann M, Eden OB, Barnes J, Pinkerton CR, on behalf of the United Kingdom Children’s Cancer Study Group (UKCCSG). MACHO chemotherapy for stage VI B cell lymphoma and B cell acute lymphoblastic leukemia of childhood. Br J Haematol 1990; 76: 359-64. 34. Barlogie B, Epstein H, Salvanayagam P, Alexanian R. Plasma cell myeloma: new biological insight and advances in therapy. Blood 1989; 73: 865-79. 35. Beisehuizen A, Hahlen K, van Wering ER, et al. Detection of minimal residual disease in childhood leukemia with the polymerase chain reaction. N Engl J Med 1991; 342: 772-74. 36. Greaves MF. Speculation on the cause of childhood acute lymphoblastic leukemia. Leukemia 1988; 2: 120-25.
VIEWPOINT
Couple screening for cystic fibrosis The main aim of screening for congenital disorders is the prevention of handicap with the least harm from side-effects (anxiety and unnecessary invasive procedures) and at the lowest cost for a given level of prevention. It is best achieved by delaying screening until the latest time when effective preventive action can be taken and by choosing an occasion when all people to be screened are likely to be accessible-at what might be termed a natural "screening turnstile", such as birth, school entry, or in early pregnancy. The outlook for patients with cystic fibrosis has been improved by the treatment of complications, but their expectation of life is still less than 25 years. There is substantial associated physical handicap’ and no known cure. An appropriate screening turnstile for cystic fibrosis is, therefore, early pregnancy. The expectant parents have the choice of antenatal screening and, if an affected fetus is identified, the option to terminate the pregnancy. Pregnancy is not only an appropriate turnstile but also a critical one, in that the result will be of immediate importance to the
expectant parents. With autosomal recessive disorders such as cystic fibrosis, only the homozygote with two allelic mutations is affected; the heterozygote with one is unaffected. Since an affected fetus can arise only when both partners each contribute a cystic fibrosis mutation, the screening unit is the couple, not the individual parent. Heterozygotes can be identified easily-all that is needed is a mouthwash sample of buccal cells for DNA analysis collected in a small tube that can be posted to the laboratory by the subject. In screening, the analysis need only be carried out if samples are received from both partners, because both are needed to determine whether a couple is at risk of having an affected fetus. However, in almost all cases only one sample (either will do) is tested, because there is no need to test the second if the first is negative. If both samples have one of the cystic fibrosis mutations tested for, the couple is designated "screen positive" and notified of a positive result; otherwise, the couple is designated "screen negative", with an option of receiving a negative report if they provide a pre-addressed envelope. All couples are, of course, informed of the procedure and the fact that screening is directed at both partners together.
Fig 1 shows the results of couple screening for cystic fibrosis and fig 2 the alternative stepwise approach, in which the mother is screened first and, if she is found to be heterozygous, the father is approached for a sample. The examples describe 250 000 pregnancies with a birth prevalence of 1 in 2500, which implies a heterozygote prevalence of 4% (4% x 4% x 0-25 = 1/2500). It is also assumed that 80% of all heterozygotes, and therefore 64% of all homozygotes (80% x 80%), can be identified. This identification rate is realistic; although more than 150 cystic fibrosis mutations have been identified, 1 (AF508) accounts for about 75% of alleles in Britain2 and another (G551D) accounts for a further 5%.3 In figs 1 and 2, a positive test is one in which a cystic fibrosis mutation is detected. The detection rate (sensitivity) is the proportion of all affected pregnancies that are positive (ie, both parents are found to be heterozygotes) and the false-positive rate is the proportion of all unaffected pregnancies that are
positive. There
important advantages of couple screening stepwise screening. (a) For the same detection rate (64%), the number of women who would be made anxious by the knowledge that they were screen positive is reduced by more than 97% (initial false-positive rate 0-08% vs 3-2%). (b) The odds that a positive initial result is a true positive increase 33-fold (64/192 or 1/3 vs 80/7920 or 1/99). (c) A definitive diagnostic test is available for all screen-positive couples (identification, in the fetus, of the same cystic fibrosis mutations as those found in the parents) which will, with the exception of rare technical errors, yield no false-positive or false-negative results. There is no definitive diagnostic test for all screen-positive women in stepwise screening. In stepwise screening, nearly every woman who is positive will have a negative partner. But even when he is shown to be negative, the position remains uncertain. The are
several
over
woman cannot
be reassured, since she is left with the
ADDRESS Department of Environmental and Preventive Medicine, Wolfson Institute of Preventive Medicine, Medical College of St Bartholomew’s Hospital, Charterhouse Square, London EC1 M 6BQ, UK (Prof N J Wald, FRCP)
1319
Fig 1-Couple screening for cystic fibrosis.
knowledge that because she is heterozygous, there is a
1 in 500 chance that the fetus is affected (80-64/7920-192); the father may have one of the mutations that was not included in the screening test. This is a low risk, but it is five times higher than the general risk of 1 in 2500. The antenatal diagnosis of cystic fibrosis in such pregnancies is difficult and subject to error. If the fetus did not have the mutation identified in the mother, it would be unaffected; but if the maternal mutation were present in the fetus, the chance that the fetus would have cystic fibrosis is about 1 in 250 when the father is screen negative. The use of the microvillar enzyme test4 on amniotic fluid would improve the discrimination. The test can detect 95% of all affected fetuses with a cutoff level that would also identify 8% of unaffected pregnancies as positive. The odds of having an affected fetus would therefore be 1 in 20, so that although the detection rate could be increased from 64% to nearly 80% (fig 2), the cost would be termination of 20 unaffected pregnancies for each cystic fibrosis pregnancy. If the microvillar enzyme test were negative, the odds would be only 1 in 4400. An advantage of couple screening is that it avoids the unnecessary worry that can arise if a woman is found to be a heterozygote but her partner is not available.
A minor disadvantage of couple screening is that samples for DNA analysis are required from both partners (even though the second of each pair is tested only when the test reveals a cystic fibrosis mutation in the first), whereas with stepwise screening they are required only from the women and from about 3-4% of men. Since the specimens needed are aqueous suspensions of buccal cells, the extra cost is negligible; the practice of obtaining samples from both partners before testing ensures that both members of the couple agree to be screened at the outset. Although the uptake may be low because couples might not take the trouble to post their samples to the laboratory, this has not been our experience. A random sample of women who inquired about serum screening for Down’s syndrome were informed about cystic fibrosis couple screening and were sent details together with sample tubes and a reply-paid package; the uptake was 75% (70/94). None of the couples screened asked to know whether the individual tested was a carrier, so inquiries about whether one or other partner is a carrier are likely to be rare. In the event that one partner may wish to know his or her carrier status, he or she would be asked to attend to discuss the request and, if necessary, to be tested. That individual might not have been the one who was tested previously. Antenatal couple screening is therefore a practical and efficient method of screening for cystic fibrosis, but it needs to be implemented on a wider scale and among other, perhaps less motivated, groups of antenatal patients before being introduced into routine obstetric care. The two existing antenatal screening procedures for serious congenital disorders are alphafetoprotein (AFP) screening for open spina bifida, and serum screening for Down’s syndrome by means of AFP, unconjugated oestriol, and human chorionic gonadotropin measurement interpreted in conjunction with maternal age. AFP screening for spina bifida has a detection rate of about 75% with a false-positive rate of 2-3%.s Serum screening for Down’s syndrome has a detection rate of 60% with a false-positive rate of 5%.6 Antenatal couple screening for cystic fibrosis has a detection rate of about 64% and a false-positive rate of 01%, so it is the most effective of the three screening programmes, achieving comparable detection rates at less than a tenth of the false-positive rate. I thank Professor Howard Cuckle and Professor David Brock for useful discussions during the development of the couple screening concept; Dr Ephraim Gazit, Professor Bob Williamson, Dr Caroline Williams, and Ms Annette Gilfilliln for technical help in introducing the DNA technology into our laboratory; Ms Lynne George for the DNA analyses; Ms Karen Fordham and Ms Anne Kennard with the other members of the antenatal screening staff in the department for organising the implementation of the screening service; and Professor Eva Alberman for commenting on the paper.
REFERENCES
Fig 2-Stepwise screening for cystic fibrosis.
1. The British Paediatric Association Working Party on Cystic Fibrosis. Cystic fibrosis in the United Kingdom 1977-85: an improving picture. Br Med J 1988; 297; 1599-602. 2. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1073-80. 3. Cutting CR, Kasch LM, Rosenstein BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide binding fold of the cystic fibrosis conductance regulator protein. Nature 1990; 346: 366-69. 4. Brock DJH, Mennie ME, McIntosh I, Jones C, Shrimpton AE. Heterozygote screening for cystic fibrosis. In: Drife JO, Donnai D, eds. Antenatal diagnosis of fetal abnormalities. London: Springer-Verlag, 1991: 59-70. 5. Wald NJ, Cuckle HS. Biochemical detection of neural tube defects and Down’s syndrome. In: Turnbull A, Chamberlain G, eds. Obstetrics. Edinburgh: Churchill Livingstone, 1989: 269-89. 6. Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum screening for Down’s syndrome in early pregnancy. Br Med J 1988; 297: 883-87.