British UedlcaJ Bulletin (1992) \PW 48, No 4, pp8O5-822 C The Bnlnt Cornell 1992
Screening for cystic fibrosis H C Ryley Department of Medical Microbiology, University of Wales College of Medicine, Cardiff, Wales
M C Goodchild Department of Child Health, University of Wales College of Medicine, Cardiff, Wales
J A Dodge The Queen's University of Belfast, Belfast, Northern Ireland
Neonatal screening for cystic fibrosis (CF) reduces short-term morbidity but its long term effects remain to be demonstrated. The best available method is the assay of immunoreactive trypsin in dried blood spots, and specificity can be improved by adding direct or indirect genetic analysis. Pregnancies known to be at risk of CF can also be screened by molecular methods, and affected pregnancies terminated. The application of genetic testing to whole communities, to detect unknown heterozygotes, raises many questions which require consideration by society and the health professions. The development of effective treatment of the basic abnormality of cell function in CF would enhance the need for neonatal screening, and possibly reduce the requirement for abortion.
Cystic fibrosis (CF) can be a difficult disease to diagnose, especially in the very young. Although the affected infant or young child may present with the relevant symptoms, such symptoms are not diagnostic and may vary, both in nature and severity. Because failure to make an early correct diagnosis has potentially serious consequences, it is not surprising that CF has been considered a prime candidate for mass neonatal screening. At the time of writing, there is no cure for CF, only management of the secondary pathology, with the aim of retarding the progressive clinical deterioration. In this situation, early identification of the affected fetuses, followed by pregnancy termination, becomes an alternative strategy for control and reduction of the disease. At present, it is practical to offer prenatal and antenatal screening procedures, with
their particular requirements for counselling and timing and which carry some hazard to the pregnancy, only for those pregnancies which are known to be at risk. This applies to couples who already have a CF child or where there is a close family history. As most CF children are the first affected children in their families, this represents only a small proportion of at risk pregnancies per year. Identification of the vast majority of CF pregnancies could be achieved by mass carrier screening—and such heterozygote screening has become feasible with the characterisation of the gene in which the CF mutations occur. This review covers the current state of tests available for neonatal, prenatal and population screening and comments on the implications—clinical, ethical and financial. NEONATAL SCREENING Ideally a neonatal screening test should be not only specific for CF, but quick, simple and inexpensive. Currently there is no test that fulfils all these criteria and compromises have to be made. The value of all screening tests depends ultimately on the precision and accuracy of the definitive test for the disease, which in die case of CF, remains the sweat test To date it has not been superseded by genetic tests, though they can be useful occasionally for confirmation if the sweat test is doubtful or equivocal. The sweat test The sweat test is based on the observation in 1953 by Darling et al.1 that stimulated sweat of CF patients contains elevated levels of sodium and chloride ions. Gibson and Cooke2 devised the most accurate method, the quantitative pilocarpine iontophoresis method. Sweat production is stimulated by pilocarpine, which is placed on the skin and directed to the sweat glands using a potential gradient (iontophoresis); the sweat is absorbed onto salt-free pads and analyzed for sodium and chloride ions by routine clinical chemical mediods. If carried out properly and with considerable care, this method is still the most specific biochemical test for CF,3 although false negative results have been reported4'5 as well as consistently borderline values.6 False positive results can also occur7 often due to lack of care during sweat collection, resulting in evaporation of collected sweat prior to analysis. Consequently the test which is time-consuming should be done only by properly trained personnel. An additional problem is that in the very young and those with dry skin, sweat collection volumes may be too small for analysis.
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There have been numerous attempts to improve, or simplify, the Gibson-Cooke sweat test, with mixed results. Although most tests still depend on pilocarpine iontophoresis for sweat stimulation, other methods of collection and ion estimation have been proposed. Chloride ion selective probes placed directly in stimulated sweat8 have proved less specific than the Gibson-Cooke method9 probably due to sweat evaporation and the meticulous care which must be taken to avoid protein build-up on the ion selective membrane (P Bray, personal communication, 1977). In 1964, Wescor Ltd introduced an alternative collecting device called the MACRODUCT Sweat collecting system. It consisted of a flexible plastic coiled capillary tube, containing a spot of blue dye at the aperture of the tube, which moves along the tube as the sweat is collected, providing a simple visual measure of the volume accumulated. Sweat analysis from these devices is usually based on osmolality and findings have been comparable with those derived by the Gibson-Cooke method10-'J despite the fact that osmolality can be influenced by nonspecific changes in sweat conditions, such as changes in lactate content Capillary collection appears to overcome the problem of evaporation but positioning and fixing die device can be a problem in the very young. A semiquantitative test, based on die production of a white silver chloride precipitate ring on a brown silver chromate background, was originally proposed by Shwachman and Gahm12 and more recently adapted to die so-called paper patch test 1 3 Although intended for the non-specialist centre, die mediod is very subjective and liable to misinterpretation and has not gained popularity. Altiiough die sweat test is die basis for clinical diagnosis of CF, neidier pilocarpine iontophoresis nor any of die modifications discussed above, is suitable for mass routine screening. The approach therefore, has been to filter out most normal children using less specific methods, and to apply the sweat test to the few who are positive on initial screening. This system is only valid if false negative results obtained by routine screening are few and if false positive results are not too frequent, in view of dieir potential to distress families. The following sections discuss mediods of routine screening. Meconium test This test is based on an observation that the albumin content of meconium from CF infants is elevated, thought to be due to the absence of pancreatic function in the CF fetus and consequent failure to digest swallowed amniotic fluid protein.14 Routine screening for meco-
mum albumin began in the mid 1970s using either a qualitative dip stick (the BM meconium test) 15 or various quantitative immunoassay methods.16- 17 It soon became clear that the method had both a relatively high false positive incidence (>0.5%) and a significant false negative incidence (>25% of all CF neonates.)18- 19 Additional procedures were proposed to increase both the sensitivity and specificity of the test by estimating also the lactase and 6-D-fucosidase activity20 or estimating pancreatic proteolytic activity in early faecal specimens.21 Although such combined or alternative tests may reduce the false positive incidence there has been no convincing evidence that they have had a major impact on the false negative incidence. Nevertheless, despite its shortcomings, meconium screening is still carried out in several centres and hospitals both in the UK and in Europe, due to its low cost and simplicity. Blood immunoreactive trypsin (IRT) screening In 1979 Crossley et al. 22 reported a two to threefold increase in IRT in blood from CF neonates, compared with normal (non-CF) infants. The test was based on a radioimmunoassay for serum trypsin, and was adapted for use on dried blood-spots. Consequently routine IRT screening for CF could be integrated easily into existing neonatal screening programmes for the detection of phenylketonuria and hypothyroidism, using dried blood-spots. The usefulness of the IRT test is limited to the neonatal period, as IRT levels begin to fall to normal within a few weeks or months of the child's birth. In most older CF children, IRT levels are subnormal but there is considerable child to child variation. Thus in our own study23 one CF child was found to have an IRT value which became subnormal within a few weeks of birth, whereas other CF children have maintained elevated IRT levels up to 3 years (Ryley, unpublished). In the initial prospective studies, the false positive incidence was 0.5% (from first blood spots) but the false negative incidence was very low if infants presenting with meconium ileus were excluded.24- 25 The false positive incidence could be significantly reduced by repeating the test on a second blood spot. More recently, Travert26 correlated the results of routine screening from 16 centres around the world. The false positive rate was 0.2 to 0.5% although this was higher in those laboratories where a lower cut-off point was taken. False negative figures were more variable from centre to centre (0 to 20%) but most laboratories reported an incidence of less than 10%. Large scale mass screening results published between 1988-91 show similar trends (Table 1).
Table 1 Results of some recent routine screening programmes for elevated IRT in dried blood spots
Positive on 1st Specimen
Positive on follow up
CF Patients Positive on Screening
CF Patients Negative on Screening
Method of Assay
Roberts et al 198827
RIA-Modified O S (Sonn) Kit
Wesley et al 198932
RIA-Modified O S (Sonn) Kit
Chatfield et al 199123
Hammond et al 199128
RIA-Modified CIS (Sonn) kit
g p point was 120 ng/ml: b Repeal test cut off point was 80 ng/ml: NR = Not Reported.
ING FOR CYSTIC FIBROS
False negative figures in particular are open to interpretation, depending to some extent on cut-off values (reducing the cut-off point may dramatically decrease false negative rates, but with an obvious rise in false positives). The false negative incidence should include all infants missed, not only those negative on the first test but also those who although positive on the first test are negative on follow-up.29- 3° Specimens from infants with meconium ileus have a high (over 30%) false negative incidence29- 3° and as such infants are all presumed to have CF, most workers exclude such specimens from their assessments of the specificity of the test. In the last analysis, all figures for false negative incidence are likely to be underestimates as account cannot be taken of long delays in clinical diagnosis, for some patients.31 In addition some infants are missed due to administrative failure.32 Recently there have been reports concerning improvement in sensitivity and specificity for the IRT test, by including the use of complementary tests. Pederzini et al. 33 have suggested a combination of meconium screening by measurement of lactase, on those infants who are IRT positive. Sweat tests are done on those patients who test positive, either by the lactase test or where blood spots are above a certain value by IRT test This approach achieved a marked drop in false negative incidence but it seems likely that the extra work and expense will be unacceptable. A combined chemical and genetic approach has been carried out by Ranieri et al. 34 in South Australia, whereby IRT positive spots were tested for delta F508 and delta 1507. Only those patients carrying either mutation (in single or double copy) were sweat tested. Patients with meconium ileus were also tested, regardless of IRT results. By these means it was possible to reduce the number of false positive tests by IRT and the number of sweat tests, and so keep parental anxiety to a minimum—only 2.8 families were contacted for every case of CF diagnosed. At the time of reporting, no cases of affected infants were known to have been missed. The system avoids the slightly increased false negative rate which comes from false negative second blood spots, found on IRT screening only. In the future, genetic screening could include other less common mutations and the exact mutations sought should be chosen according to their relative frequency in die population being screened. Naturally the genetic screening will recognise CF heterozygotes as well as homozygotes and in the above study genetic counselling was provided for all families contacted. A slightly different 2-tier approach, using die CF associated KM 19 restriction site marker rather than direct gene analysis was used by Laroche and Travert 35 This was particularly useful in the Normandy population, where 22% of CF patients do not have the delta F508 deletion on either chromosome. 55% of the hypertrypsinaemic infants
SCREENING FOR CYSTIC FIBROSIS
homozygous for the KM 19 allele 2 had CF whereas the risk was reduced to less than 1% in those homozygous for allele 1 and to only 4% in those heterozygous for both alleles. If homozygotes 1-1 had not been recalled for sweat testing, the false negative rate would increase from 4 to 6%. In the Normandy population, detection of the delta F508 deletion gives worse results than KM 19 in terms of sensitivity (20% of CF patients would be missed) and is significantly more expensive. The value of neonatal screening The cost of the IRT test, based on our own experience is summarised in Table 2. We estimate it to be about £5000 per child diagnosed, using IRT screening. This cost excludes the collection of the blood spot because we assume any such CF screening would be integrated into established screening systems for phenylketonuria (PKU) and hypothyroidism. The cost compares with established screening systems for phenylketonurea (£7,700) and hypothyroidism (£4,000) in Wales (D Bradley, personal communication). Unfortunately, unlike PKU we are only beginning to understand the biochemical and genetic defects that cause CF—although it seems increasingly likely that there will be an effective genetic or pharmacological treatment for CF in the foreseeable future. Current treatment regimes, directed against the secondary pathology of the disease, have been effective to a degree—as shown by the improved prognosis over the past 20 years, reported by the BPA Working Party on CF.36 It could be argued that results should be even better if the diagnosis were made and treatment started earlier, but this remains to be shown. Several centres have attempted to address this matter. Unfortunately some studies have been concerned with longitudinal comparisons of screened and unscreened; that is, an examination of CF babies recognised through an IRT screening programme and those diagnosed in earlier years, on clinical grounds. Results have been mixed. Wilcken and Chalmers37 compared screened and unscreened infants up to 2 years of life, the only significant difference being that the unscreened infants spent longer in hospital, for CF-related illness, implying that screening reduces morbidity during these early years. Bowling et al.38 made a similar comparison to the age of 2 years, and found fewer chest infections and a better weight gain in the screened group. Dankert-Roelse et al 39 conducted a longer follow-up on Dutch children, with the second evaluation at 9 years. Although not all these children were treated in an exactly comparable manner during their early years (more of the screened patients being managed at a 'CF Centre') nevertheless the children were all born during the same time
Table 2 Estimate of the cost of routine IRT screening based on our experience in Wales Material Cost of IRT Test for one year in Wales Based on 40 000 specimens and including reagents (Behnng RIA kit) tubes and repeat specimens
Technician's Salary (MLSO Grade 1>+ 22%
Cost of Equipment (gamma counter and centrifuge) depreciation including service when required Estimation is based on expected 10 year life of the equipment
Cost of services to shared room (based on UHW, Cardiff estimate)
Cost of sweat testing screened positive infants (20 per year) Total cost Cost per child diagnosed based on an expected detection of 10 CF infants annually (this excludes infants with meconium ileus and those giving a false negative screening result)
£200 £45 250
span. Results from these studies were more conclusively in favour of screening: these patients had significantly better clinical and chest X-ray scores; serum values for total IgG and vitamin A were higher for the screened, who showed a better rate of survival. However none of the lung function test measurements differed between the 2 groups. In an effort to carry out a larger survey which would be a concurrent evaluation of screened and unscreened, a 5-year (1985-1989) study by IRT was set up in Wales and the West Midlands, UK and follow-up continues.23 Infants were screened during alternate weeks in each region. Management is by the local paediatricians or the regional centre according to normal referral patterns, via a suggested protocol. A range of data is obtained annually including anthropometry, Shwachman and chest X-ray scores, routine microbiology, haematology and biochemistry. Hospital admissions are recorded. Analysis of results to 4 years shows a longer hospital admission time (excluding time for diagnosis of CF) for the unscreened during the first year, but no other significant differences. There were significantly fewer children 'not at risk' (ie who did not present with meconium ileus or have a sibling with CF) in the unscreened group; thus this group may be lacking children still undiagnosed or who have died without the diagnosis being made. Further follow-up and the evaluation of respiratory function tests should show whether the screened and unscreened groups diverge later on.
SCREENING TOR CYSTIC FIBROSIS
While clinical benefits gained by screening remain controversial, there are other, undoubted advantages of early diagnosis. The identification of a 'CF family' while the affected child is still an infant allows considered decisions regarding further children and may help to allay the feelings of anxiety, guilt and discontent with the medical services which a prolonged search for a diagnosis can engender.40 Without screening (or early diagnosis) there are many cases where an elder child with CF is not diagnosed until a younger sibling has been detected, so inflicting a double tragedy on the family, but parents of a CF child rate the psychological and emotional advantages of screening higher than its value for genetic counselling.40- 41 The contrary argument—that parents of false positive infants incorrectly suspected of having CF on the first IRT test suffer needless anxiety—has been used against CF screening. In a Dutch study such parents, while undoubtedly wishing they had not had such a traumatic experience, nevertheless supported the aims of die programme and regarded its effect as on balance beneficial. The authors of the report emphasized the importance of minimal delay between die screening test and the definitive sweat test42 as have others.40 Although the definitive treatment for the basic defect may still be some way into the future, such treatment will be almost certainly more effective for children with the minimum of complications—a situation which could be governed by early diagnosis. If this is so, prenatal screening or at least early neonatal diagnosis, will be necessary. It would clearly be advantageous to have a proven effective service in place prior to this time. PRENATAL SCREENING Prenatal diagnosis of cystic fibrosis is an important option in the genetic counselling of parents but it has been less than 10 years since such diagnosis could be given with any confidence. The first reliable test was based on the discovery that the activity of some of the rmcrovillar membrane enzymes in amniotic fluid was depressed if the fetus had CF.43> ** There were some problems associated with the test. Its reliability depended on measuring enzyme activity at an optimum gestation time of 17-18 weeks, so that termination, if appropriate, was late in the pregnancy and often not possible before 20 weeks. There are difficulties also in assessing the reliability of the test, especially the false positive incidence, as fetuses that gave a positive result rarely went to term. However, Boue et al. 45 carried out an examination of such fetuses formulating criteria that might be associated with CF. In all cases examined, each fetus that gave a positive result fulfilled at least one of
these criteria. Overall, for pregnancies with a 1:4 risk, the false negative rate was 5% and the false positive rate 8%.46 The discovery of DNA markers tightly linked to the CF locus 47 - 49 lead to the use of such markers for prenatal diagnosis by 'linkage'. 50 This was a major advance, allowing prenatal diagnosis by testing of a chorionic villus sample (CVS) obtained by biopsy (usually transabdominal) during the first trimester at about 10 weeks of gestation; also a greater accuracy of prediction and earlier termination if necessary, by 12 or 13 weeks. 'Markers' are DNA polymorphisms, or more correctly called 'restriction fragment length polymorphisms' (RFLPs) which are situated close to the gene in question and used for gene tracking; they are detected by 'probes' which are radiolabelled DNA fragments. The linkage test for antenatal diagnosis of CF depends on a number of factors. First, close linkage, or a very short genetic distance between marker and gene, means that recombination during meiosis, between marker and gene, is very unlikely. Second, the polymorphism of the markers and the distribution of the alleles within the nuclear family permits a distinction to be made between 'CF' and 'non-CF' chromosomes. Finally, it is essential that DNA is available from the index case so that 'phase' can be established—that is, the association between the CF chromosome and its marker alleles. Now that a larger number of DNA markers can be detected, on either side of the CF gene, virtually all CF families can be informative, using this method. It is necessary sometimes to employ up to 6 markers or even more to deduce phase from the combined results, as some markers individually may be uninformative (ie do not allow tracking of the CF gene within the family).51. 5 2 Examples of problems with tracking of the CF gene, using a linked marker, and successful tracking, using one or more markers, are shown in Figures 1 and 2 Accuracy of antenatal diagnosis of CF by linkage has increased rapidly over the last few years so that the potential error from recombination is now essentially negligible.46 The location and characterisation of the CF gene itself53 has transformed the situation yet again. The need for linkage studies and enzyme tests has been further reduced. Antenatal diagnosis is still done by chorionic villous biopsy at 10 weeks of gestation, but with the ability to identify individual mutations within the CF gene, rather than utilising the markers flanking the gene, diagnosis has become even more reliable. PCR methods allow diagnosis within 1-2 days. For specific mutations which can be identified, accuracy should be 100%. Although it is still advisable to take blood for DNA from the index case soon after birth (in case linkage studies should be required later) it is no longer necessary, if a specific gene mutation can be recognised in both
SCREENING FOR CYSTIC FIBROSIS
1-1 No surviving affected child
Everybody homozygous for this probe
Everybody heterozygous for this probe
50% chance fetus is affected 50% chance it is a carrier
Fig. 1 Problems in tracking the cystic fibrosis gene using a linked marker. (Reproduced with permission from papeT by A P Read in J R Soc Med 1987, 80 (Suppl 15) 11-15)
parents, for affected family members to be alive or their DNA stored. Antenatal diagnosis can be applied reliably to pregnancies where both parents have been shown to be carriers, so there is a 1:4 risk. Other family members, such as second and third degree relatives, can benefit also from carrier screening. Spouses of known carriers can be tested for the commoner CF mutations. If these results are negative the risks of the couple conceiving CF children are substantially reduced.538 It has become possible to combine prenatal screening with in vitro fertilisation and to screen the zygote prior to implantation but only one selected pregnancy has been achieved so far, from a few CF families, by this method (RML Winston, personal communication).
B l_l— 2-1
Fetus homozygous normal
Fetus homozygous normal oir carrier
I 1 A2-1 B1-1
Fetus homozygous normal
Fig. 2 Examples of successful tracking of the cystic fibrosis gene using a linked marker (Reproduced with permission from paper by A P Read in J R Soc Med 1987; 80 (Suppl 15). 11-15).
The majority of Regional Genetic laboratories now analyze for the most common CF mutation, delta F508, and 5-10 other mutations; taken together these tests will identify about 85% of CF patients. It would be impracticable for a laboratory to test for all the 150+ CF mutations now known to exist: time and cost would be prohibitive and many of these mutations have been reported in single families only. The mutations selected for test in any particular region will depend on prior knowledge of those most common within the local population. Therefore despite the recognition of the CF gene and its many mutations, there are still circumstances when antenatal diagnosis cannot be done by direct gene analysis. It is not uncommon for one parent in a CF family to carry an unidentifiable CF mutation; much less common is the situation where neither parent carries an identifiable mutation. For both these types of families, linkage studies are still necessary. If a mother with a family history of CF with an unknown genotype presents late in pregnancy, ie during the second trimester, then the microvillar enzyme test is still relevant, possibly combined with an examination of amniotic fluid cells for the CF allele(s). This combination of tests, with limited accuracy, could be offered also to some families of unknown genotype where the CF index child has died without DNA being stored and to certain families with a low prior risk of CF but with a high degree of anxiety. Acceptance of antenatal diagnosis of CF, with a view to abortion, has varied from 20% of couples interviewed54 through 52% 40 to 65% 55
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Many factors have an influence54 but neither educational standard nor social class has been implicated.40 Ideally, genetic counselling should be done before another pregnancy is underway, to allow a calm appraisal of all the issues.56 As more information becomes available, the issues are more complex and counselling should be done by personnel who are thoroughly familiar with the disease and its ramifications. Only time will tell whether certain issues relating to recent research should be included in genetic counselling, for example whether there are meaningful correlations between CF phenotype and genotype: so far such correlations appear to be confined to the relatively small number of patients who are pancreatic sufficient57 and to occasional patients with unusual genotypes.58 Overall, these individual variations are probably overshadowed by the multiple disease expressions found within the largest genetically 'homogeneous' group—that is the 53% or so of the CF population in Northern European countries who are homozygous positive for the delta F508. The clinical care offered to patients may be a more important determinant of their prognosis than their genotype. Antenatal diagnosis of CF is carried out only if a risk is known to exist. To detect the 80% of newly diagnosed CF patients born into families without a CF history, heterozygote population screening would be required. HETEROZYGOTE SCREENING The discovery of the CF gene and its most common mutation in 1989, has made heterozygote population screening a possibility.59 Although the delta F508 mutation accounts for no more than 75% of carriers in most Northern European populations, the detection rate can be increased by concurrent identification of a small number of other, much less common mutations, so that the percentage of mutations detected rises to about 85%.46-