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Screening for cystic fibrosis carriers
984
We also verified sensitivity by mixing the sample from a known AF508 heterozygote with 4,9,14, and 19 samples from non-AF508 controls. In all the assays with multiple samples, the heteroduplexes formed by cross-hybridisation of the 95 and 98 base pair (bp) PCR products could be detected, although the 95 bp fragment was not visible. AF508 homozygotes could be detected, since heteroduplexes between the 95 bp patient’s product with the 98 bp fragments from other samples also appeared, giving a false
heterozygous reading. The theoretically optimum number of samples to be pooled initially depends on the expected prevalence of AF508 heterozygositY’ (19 if carrier probability is 1/28 or 31 if carrier proability is 1/45). However, pooling more than 20 samples in the same assay would require a higher reaction volume and may increase the risk of false negatives. The advantage of this procedure is simplicity and reduction in the cost and work required for AF508 screening. Hundreds of samples can be analysed in a day. The use of blood spots on filter paper, avoiding the DNA isolation step, decreases the risk of sample contamination and may increase uptake by facilitating the collection of samples, which could be done by individuals at home and mailed to the laboratory in sterile plastic bags. The pooling strategy reduces 5 to 7 times the number of assays needed. This procedure would also be suitable for neonatal CF screening since both AF508 homozygotes and heterozygotes can be detected.
Supported in part by Cilag (Johnson & Johnson) Laboratories. Department of Paediatrics, Hospital Infantil Niño Jesús, 28009 Madrid, Spain
J. C. MOLINA CABAÑERO L. A. PEREZ JURADO
Division of Genetics, Vanderbilt University,
Nashville, Tennessee, USA
S. RASKIN
Histocompatibility Unit,
J. L. VICARIO A. BALAS PEREZ M. D. GARCIA NOVO
Transfusion Centre, Madrid
ME, Gilfillan A, Compton M, et al. Prenatal screening for cystic fibrosis Lancet 1992; 340: 214-16. Watson EK, Mayall ES, Lamb J, et al. Psychological and social consequences of community carrier screening programme for cystic fibrosis. Lancet 1992; 340: 217-20. Statement from the National Institutes of Health workshop on population screening for the cystic fibrosis gene N Engl J Med 1990; 322: 70-71. Beaudet A, O’Brien W. Advantages of a two-step laboratory approach for cystic fibrosis carrier screening. Am J Hum Genet 1992; 50: 439-40 Keren BS, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1073-80. Gille Ch, Grade K, Coutelle Ch. A pooling strategy for heterozygote screening of the &Dgr;F508 cystic fibrosis mutation. Hum Genet 1991; 86: 289-91.
1. Mennie 2.
3. 4. 5. 6.
Screening for cystic fibrosis carriers SIR,-Dr Super and colleagues (Aug 22, p 490) draw attention to the apparent merits of cascade testing for carriers of the cystic fibrosis (CF) gene. They claim that they can identify more heterozygotes with less effort by testing relatives of affected probands than my colleagues and I can through the type of population screening programme we described (July 25, p 214). This is self-evident. However, there are limits to how far cascade testing can penetrate the population. Additionally, whether cascade identification of a carrier is of any benefit is not clear, since carrier status is only important in the context of reproduction. We began to offer cascade testing in Scotland as soon as the CF gene was cloned in 1989. The take-up rate was fast at first, but then slowed, and we are now approaching a plateau at less than 1 % of the estimated 200 000 Scottish CF carriers. Even in a population as homogeneous as this one, to identify more than a minute proportion of carriers by family extension studies will not be possible. The proportion will obviously be lower in communities that have seen much immigration. The argument for population screening is thus strengthened by the failure of the cascade approach. Either we ignore the 99% of carriers who do not have an affected relative, which is what Super seems to be saying, or we try and reach them by more broad-based techniques. We have presented evidence that carrier screening for
CF during pregnancy is probably the most efficient way of doing this. We concede that it does cause transient anxiety among a few of those identified as carriers. But we believe that this is an acceptable price to pay for a form of preventative medicine that lacks the nepotistic features of cascade testing. Human Genetics Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
D.
J. H. BROCK
Cystic fibrosis mice with disease-related changes in lung and reproductive tract SIR,-In your Sept 19 editorial you comment on mice homozygous for cftr gene disruption. These mice were produced concurrently and independently by three groups using gene targeting in embryonal stem cellos. 1-3 You report that most mutant mice die perinatally or at weaning because of intestinal obstruction, and that, in contrast to cystic fibrosis in man, no excess mucus is seen in the lung and the male reproductive tract is normal. You
highlight the failure of the mice to mimic important hallmarks of the disease (notably lung and gonadal involvement) and suggest that the usefulness of these laboratory animals will be increased if the intestinal disorder can be alleviated. To describe the homozygous cftr mutant mice from all three groups as having a single phenotype is misleading. The distinct phenotype of our micemerits further comment.
Firstly, our cystic fibrosis mice do not die perinatally. Most of our homozygous mutant mice (cflcj)survive weaning, and cannot be distinguished visually from their wild type (+ / +) and heterozygote (cfl + )littermates at 30 days. However, postmortem examination of 4 of 6 c :fl cf mice showed overt gastrointestinal pathological changes, including abnormal mucus accumulation. Mild dilation of the salivary ducts was also seen. Most importantly, 1 mouse showed clear evidence of mucin in the lung, causing mild focal atelectasis consistent with presymptomatic lung disease. 1 of 2 male mice examined showed gross accumulation of mucin in the vas deferens. We have suggested two possible explanations for the phenotypic differences between our homozygous mutant mice and those described by the Chapel HilP and Cambridge2 groups. The first is that our mutation was crossed onto an outbred genetic background whereas the cftr mutations in the other two studies were crossed onto inbred strains. It is noteworthy that comparable cystic fibrosis mutations arising in ethnically closed human populations are severe whereas those arising in ethnically mixed populations are typically mild. A second possiblity is that a trace amount of residual normal cftr protein may result from aberrant splicing of our insertional targeted mutation, whereas this could not occur in a cftr gene disrupted by targeted gene replacement. A low level of wild-type protein in our insertional efl ef mice might explain why they do not show the extreme degree of intestinal disorder that is seen in both the Chapel Hill and Cambridge replacement mutant mice. Any wild-type protein present must be at a very low level, because the expected chloride channel defect is demonstrable by in-vivo potential differences in all our insertional cflcf mutant mice.3 Further experiments are necessary to resolve this issue. In any event, the enhanced viablity of our cystic fibrosis mouse model uniquely allows the development of aspects of the disease more relevant to the human condition. Thus our insertional cftr mutant mice constitute a valuable model to explore the pathophysiology of the disease, the relation between genotype and phenotype, and the development and testing of therapies relevant to the treatment of
patients. 3.4 MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK
JULIA R. DORIN DAVID J. PORTEOUS
1. Snouwaert JN, Brigman KK, Latour AM, et al. An animal model for cystic fibrosis made by gene targeting. Science 1992; 257: 1083-88. 2. Colledge WH, Ratcliff R, Foster D, Evans MJ. Cystic fibrosis mouse with intestinal obstruction. Lancet 1992, 340: 680. 3. Dorin JR, Dickinson P, Alton EWFW, et al. Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 1992; 359: 211-15. 4. Wilson JM, Collins FS. More from the modellers. Nature 1992; 359: 195-96
We also verified sensitivity by mixing the sample from a known AF508 heterozygote with 4,9,14, and 19 samples from non-AF508 controls. In all the assays with multiple samples, the heteroduplexes formed by cross-hybridisation of the 95 and 98 base pair (bp) PCR products could be detected, although the 95 bp fragment was not visible. AF508 homozygotes could be detected, since heteroduplexes between the 95 bp patient’s product with the 98 bp fragments from other samples also appeared, giving a false
heterozygous reading. The theoretically optimum number of samples to be pooled initially depends on the expected prevalence of AF508 heterozygositY’ (19 if carrier probability is 1/28 or 31 if carrier proability is 1/45). However, pooling more than 20 samples in the same assay would require a higher reaction volume and may increase the risk of false negatives. The advantage of this procedure is simplicity and reduction in the cost and work required for AF508 screening. Hundreds of samples can be analysed in a day. The use of blood spots on filter paper, avoiding the DNA isolation step, decreases the risk of sample contamination and may increase uptake by facilitating the collection of samples, which could be done by individuals at home and mailed to the laboratory in sterile plastic bags. The pooling strategy reduces 5 to 7 times the number of assays needed. This procedure would also be suitable for neonatal CF screening since both AF508 homozygotes and heterozygotes can be detected.
Supported in part by Cilag (Johnson & Johnson) Laboratories. Department of Paediatrics, Hospital Infantil Niño Jesús, 28009 Madrid, Spain
J. C. MOLINA CABAÑERO L. A. PEREZ JURADO
Division of Genetics, Vanderbilt University,
Nashville, Tennessee, USA
S. RASKIN
Histocompatibility Unit,
J. L. VICARIO A. BALAS PEREZ M. D. GARCIA NOVO
Transfusion Centre, Madrid
ME, Gilfillan A, Compton M, et al. Prenatal screening for cystic fibrosis Lancet 1992; 340: 214-16. Watson EK, Mayall ES, Lamb J, et al. Psychological and social consequences of community carrier screening programme for cystic fibrosis. Lancet 1992; 340: 217-20. Statement from the National Institutes of Health workshop on population screening for the cystic fibrosis gene N Engl J Med 1990; 322: 70-71. Beaudet A, O’Brien W. Advantages of a two-step laboratory approach for cystic fibrosis carrier screening. Am J Hum Genet 1992; 50: 439-40 Keren BS, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1073-80. Gille Ch, Grade K, Coutelle Ch. A pooling strategy for heterozygote screening of the &Dgr;F508 cystic fibrosis mutation. Hum Genet 1991; 86: 289-91.
1. Mennie 2.
3. 4. 5. 6.
Screening for cystic fibrosis carriers SIR,-Dr Super and colleagues (Aug 22, p 490) draw attention to the apparent merits of cascade testing for carriers of the cystic fibrosis (CF) gene. They claim that they can identify more heterozygotes with less effort by testing relatives of affected probands than my colleagues and I can through the type of population screening programme we described (July 25, p 214). This is self-evident. However, there are limits to how far cascade testing can penetrate the population. Additionally, whether cascade identification of a carrier is of any benefit is not clear, since carrier status is only important in the context of reproduction. We began to offer cascade testing in Scotland as soon as the CF gene was cloned in 1989. The take-up rate was fast at first, but then slowed, and we are now approaching a plateau at less than 1 % of the estimated 200 000 Scottish CF carriers. Even in a population as homogeneous as this one, to identify more than a minute proportion of carriers by family extension studies will not be possible. The proportion will obviously be lower in communities that have seen much immigration. The argument for population screening is thus strengthened by the failure of the cascade approach. Either we ignore the 99% of carriers who do not have an affected relative, which is what Super seems to be saying, or we try and reach them by more broad-based techniques. We have presented evidence that carrier screening for
CF during pregnancy is probably the most efficient way of doing this. We concede that it does cause transient anxiety among a few of those identified as carriers. But we believe that this is an acceptable price to pay for a form of preventative medicine that lacks the nepotistic features of cascade testing. Human Genetics Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
D.
J. H. BROCK
Cystic fibrosis mice with disease-related changes in lung and reproductive tract SIR,-In your Sept 19 editorial you comment on mice homozygous for cftr gene disruption. These mice were produced concurrently and independently by three groups using gene targeting in embryonal stem cellos. 1-3 You report that most mutant mice die perinatally or at weaning because of intestinal obstruction, and that, in contrast to cystic fibrosis in man, no excess mucus is seen in the lung and the male reproductive tract is normal. You
highlight the failure of the mice to mimic important hallmarks of the disease (notably lung and gonadal involvement) and suggest that the usefulness of these laboratory animals will be increased if the intestinal disorder can be alleviated. To describe the homozygous cftr mutant mice from all three groups as having a single phenotype is misleading. The distinct phenotype of our micemerits further comment.
Firstly, our cystic fibrosis mice do not die perinatally. Most of our homozygous mutant mice (cflcj)survive weaning, and cannot be distinguished visually from their wild type (+ / +) and heterozygote (cfl + )littermates at 30 days. However, postmortem examination of 4 of 6 c :fl cf mice showed overt gastrointestinal pathological changes, including abnormal mucus accumulation. Mild dilation of the salivary ducts was also seen. Most importantly, 1 mouse showed clear evidence of mucin in the lung, causing mild focal atelectasis consistent with presymptomatic lung disease. 1 of 2 male mice examined showed gross accumulation of mucin in the vas deferens. We have suggested two possible explanations for the phenotypic differences between our homozygous mutant mice and those described by the Chapel HilP and Cambridge2 groups. The first is that our mutation was crossed onto an outbred genetic background whereas the cftr mutations in the other two studies were crossed onto inbred strains. It is noteworthy that comparable cystic fibrosis mutations arising in ethnically closed human populations are severe whereas those arising in ethnically mixed populations are typically mild. A second possiblity is that a trace amount of residual normal cftr protein may result from aberrant splicing of our insertional targeted mutation, whereas this could not occur in a cftr gene disrupted by targeted gene replacement. A low level of wild-type protein in our insertional efl ef mice might explain why they do not show the extreme degree of intestinal disorder that is seen in both the Chapel Hill and Cambridge replacement mutant mice. Any wild-type protein present must be at a very low level, because the expected chloride channel defect is demonstrable by in-vivo potential differences in all our insertional cflcf mutant mice.3 Further experiments are necessary to resolve this issue. In any event, the enhanced viablity of our cystic fibrosis mouse model uniquely allows the development of aspects of the disease more relevant to the human condition. Thus our insertional cftr mutant mice constitute a valuable model to explore the pathophysiology of the disease, the relation between genotype and phenotype, and the development and testing of therapies relevant to the treatment of
patients. 3.4 MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK
JULIA R. DORIN DAVID J. PORTEOUS
1. Snouwaert JN, Brigman KK, Latour AM, et al. An animal model for cystic fibrosis made by gene targeting. Science 1992; 257: 1083-88. 2. Colledge WH, Ratcliff R, Foster D, Evans MJ. Cystic fibrosis mouse with intestinal obstruction. Lancet 1992, 340: 680. 3. Dorin JR, Dickinson P, Alton EWFW, et al. Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 1992; 359: 211-15. 4. Wilson JM, Collins FS. More from the modellers. Nature 1992; 359: 195-96