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Screening Newborns for Hereditary Metabolic Disease
Screening Newborns for Hereditary Metabolic Disease CHARLES R. SCRIVER, M.D.
Many people regard mass screening for metabolic disease as an uninspired form of endeavour. One might therefore ask: Why is there a clinic in this volume devoted to the topic? One might also question why committees are convened, dissertations published, and mass screening programmes inaugurated, when the apparent objective is to find but one baby with a biochemical imbalance of hereditary or acquired origin, amongst the many thousands of normal infants. Advances in medical knowledge have brought contagion and undernutrition under control on our continent. The legacy of these advances has been a relative increase in the case load of the chronic, degenerative and heritable diseases. 31 Consequently the community has aligned its efforts, in order to bring these "new" problems under control. Since a heritable metabolic disease may produce its effect on the patient from the first day of life, it is the consensus that the earliest possible detection of such an abnormality is desirable. Mass screening in the neonatal period is one means by which this can be accomplished. The community, however, would not be interested in mass screening for metabolic diseases unless there were definite advantages to be accrued. There are three desirable results expected from mass screening:33 1. It provides, by means of an approximate and simple test, a method to segregate those who do have a disorder from those who do not. 2. Through early detection, appropriate therapy, when available, can be initiated before the disorder incapacitates the patient. 3. When the natural history of a specific disorder is poorly understood, mass screening can supply the data for better knowledge about the condition. Such programmes may have a fourth characteristic, particularly in reference to the study of rare, hereditary metabolic diseases. An ongoing screening project may provide new knowledge which will improve the screening procedure itself.
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It seems almost a matter of fact, therefore, that mass screening for metabolic disease should be instituted as soon as possible. Caution is nevertheless indicated, for there are many questions about screening, still requiring answers. Our knowledge of the exact role for mass screening in the control of metabolic disease is far from complete, even in such a well known condition as phenylketonuria. 18 The combined interest and efforts of pediatricians, obstetricians, public health authorities, biochemists and government representatives, as well as others, will be needed to solve many of the problems. The purpose of this clinic will be to examine, both in prospect and for the record, the role of mass screening in detection and study of the rare infant with genetically determined, biochemical imbalance. The problems and decisions in this field are in the hands of today's investigators. What is happening in this field now is anything but pedestrian, and because the community is so interested in what we are doing, there is unequaled opportunity to learn a great deal, and to do much that is good.
BIOLOGICAL PRINCIPLES UNDERLYING SCREENING FOR BIOCHEMICAL IMBALANCE OF HEREDITARY ORIGIN
Mass screening for metabolic disease relies on simple chemical tests, which must also be efficient, sensitive and reliable. For the moment, let us consider the nature of the biological problems being examined by these tests. Mutation will alter the information in the gene, and, subsequent to its translation, the initial cytoplasmic protein product will also be modified. Ultimately, the transformation and transduction of energy in the cell, dependent on that protein, may also be altered. The phenotype for the mutation may therefore be detectable as a modified protein geneproduct, or it may be identified only in the effect of this modification on cellular composition and activity. Screening methods may attempt to assess directly the protein constituents within cells (e.g. in red blood cells), or the proteins which are released from the cells into body fluids. Alternatively, the screening method may evaluate modification in the composition of the extracellular fluid, particularly plasma and urine. Figure 56 summarizes schematically the opportunities to detect the results of mutations affecting apoenzymes and other cellular proteins (site 1, Fig. 56), or affecting membrane transport processes (site 2, Fig. 56). Potential sources of the sample (A, B, C) for analysis are also indicated in the scheme. The analytical procedure which will identify the mutant phenotype must be simple for use in mass screening programmes; by similar token, collection of the biological sample to be tested must also be simple.
SCREENING NEWBORNS FOR HEREDITARY METABOLIC DISEASES
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Figure 56. Schematic presentation of sites at which mutations might modify metabolism in a cell: 1, apoenzyme (E); 2, membrane transport (S -? S'). This might be identified by examining cytoplasmic proteins (A) themselves or the results of their modification of biochemical composition of plasma (B) and urine (C). S, Extracellular substrate; S', intracellular substrate; P', product formed from action of E and S'.
This, as we shall see, becomes an important consideration in screening of body fluids in the newborn infant. Blood and urine are the fluids most commonly used for mass screening of metabolic disease. Which one is chosen is dependent on precise knowledge of the kidney's role in biochemical homeostasis. Compounds with low renal clearance will be detected preferably in plasma. Compounds poorly transported by the tubules, from the glomerular filtrate, or secreted into the tubule lumen will be screened preferably in the urine. Heritable disturbances of membrane transport, phenotypically expressed in renal tubule cells, can be detected easily in urine, but only with poor reliability in plasma.
ATTITUDES AND PROBLEMS
Because there are so many problems pertaining to mass screening, a number of these will be discussed under separate headings. An overall perspective on current attitudes should be maintained, however, and the reports of two committees are recommended reading. One committee 19 analyzed the particular problems in screening for phenylketonuria; the other30 considered the broadest possible franchise for screening in the neonatal period (the text of the latter report is included in its entirety in the Appendix).
Who Should Be Screened? High-Risk Groups: A high-risk group comprises relatives of known patients with hereditary metabolic disease. Screening is most efficient
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when a proband has already been identified in a pedigree, and a search is made for other existing patients in that pedigree, or for new patients born into it. The methods used to find the high-risk patients are frequently chosen for their specificity in detecting the biochemical phenotype of the disease in question. The Population at Unpredicted Risk: This population offers the principal challenge to mass screening programmes. For this discussion the considerations are restricted to newborn infants, and in the present context this includes those infants still available for the first postnatal health examination. Obviously all newborn infants could be screened with considerable ease, because in North America most infants are born in hospital. This attitude is only partially acceptable, however, since it tends to exclude from mass screening programmes organized for hospital use those infants born at home. The problem is of significance for some of our rural communities, and particularly for other countries with obstetrical practices which are different from our own. What Diseases Should Be Screened? Screening is undoubtedly worth while for heritable metabolic disease with the following characteristics: (a) The natural history of the disease is well understood, and with few exceptions it can be reliably detected, early in life. (b) The disease, once identified by a screening procedure, can be confirmed by specific methods. (c) A therapeutic regimen is known which will alter the course of the untreated disease. (d) The therapeutic resources are easily available to the patient. Galactosemia 16 and phenylketonuria2o are examples of heritable metabolic disease with these attributes, which warrant mass screening for their early detection at the present time. 8 Alternatively, there are many diseases which do not fulfill all the criteria for mass screening. In that case, is there a reason not to screen for them? The following example illustrates this problem: The sodium content of sweat can be determined by a sodiumresponsive glass electrode applied to skin,u This method is easily applicable in the nursery, the determination requiring about three or four minutes for each infant. Data are presented in Table 36 which indicate that infants presenting with meconium ileus as a manifestation of fibrocystic disease can be distinguished from the normal infant in the first days of life. Whether all the infants who ultimately will be affected by fibrocystic disease can be discovered by this technique during the neonatal period will be determined only by further study. If it is found that infants with fibrocystic disease are detectable in the neonatal period, is one still justified in recommending mass screening of newborn infants for fibrocystic disease? Mass screening would be valid if there were an
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Table 36.
Sodium Content oj Sweat in the Newborn (Glass Electrode Technigue: l1 mEg./L.)
DAY OF
NUMBER OF
LIFE
PATIENTS
1. ....... 100 2 ........ 100 3 ........ 99 4 ........ 100 5 ........ 87 6. . . . . . .. 10 12 ....... .
MEAN do S.D.
16.10 15.97 15.91 15.80 15.46 14.92
do do do do do do
5.82 3.97 4.17 4.17 4.04 3.04
ill
MECONIUM ILEUS PATIENTS
52.1 87.7 95.7 130.6
2
3
98.1 83.4
55.0
4
61.2 75.2 92.4
Unpublished data supplied by R. B. Goldbloom, P. Sekelj and D. Cameron from (1) 2511 testings taken from 5 sites (palms and dorsa of both hands, and forehead) in 100 healthy babies (58 male, 42 female) with mean birth Weight 3480 gm. (2) Four infants with proved meconium ileus.
unequivocal therapeutic advantage conferred on the fibrocystic infant, detected in the first week of life and treated accordingly, over the undetected, untreated fibrocystic infant. The data which will decide this issue are not yet available, because the role of early prophylactic therapy in fibrocystic disease has not been defined; the answers may be found through pilot projects using this new screening technique. When to Perform the Screening Test The answers to this question also depend on clear understanding of the phenotypic manifestations, and the course of the disease. In diseases which are detectable very early in life and which have serious consequences (e.g. galactosemia, phenylketonuria or maple syrup urine disease10 ) the earliest possible opportunity for screening is warranted, since delay in therapy usually results in devastation to the patient. In other cases there may be no apparent urgency for screening if the disease is benign; and again a disease whose phenotype becomes apparent only later in life cannot be detected in the neonatal period. Wilson's disease and cystinosis are examples of this type of metabolic disease. The natural course of the disease is not the only determinant of the optimum time for screening. The patterns of medical care for the newborn infant are also important; for example: 1. Tests for a modification of the biochemical composition of the body fluids must be performed when the imba:lance has become manifest. Dietary intake of the affected substance and postnatal age of the baby will both influence the degree of imbalance. 2. Very early discharge of the infant from the nursery may preclude reliable screening in hospital. The screening programme may have to be devised to reach the child at a later age. Screening may therefore be indicated on more than one occasion in the same infant. Present-day programmes usually operate during the
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first week of life at. the time the infant is discharged from the nursery, and again at the end of the first month of life.
METHODS AVAILABLE FOR LARGE·SCALE SCREENING PROGRAMMES
The analytical phase of the screening technique must be simple, efficient, sensitive and reliable. Whenever possible, it is preferable to use a method which will provide information on many diseases from a single biological sample. By comparison, a specific test for a single disease is uneconomical for screening populations in which there are many different hereditary diseases to be found. Collection of the sample must also be a simple procedure. Screening programmes for metabolic disease in older patients have often relied on urine collected in volume; but this can be a formidable procedure in the neonatal period or with an uncooperative patient. Several different approaches have been developed for collecting the sample and for identifying a mutant biochemical phenotype. 1. Berry2 has devised a simple method whereby urine is collected on a piece of filter paper inserted in the diaper. The dried urineimpregnated paper is then subjected to a variety of chemical spot tests. An aliquot of the urine may also be eluted from the paper and chromatographed in appropriate solvent systems. The advantages of this system include simple collection of the sample and a broad spectrum of tests. Any disease which will modify the composition of urine is potentially detectable by this method. The disadvantages are primarily technical. Soiling of the filter paper by feces may occur. The necessity to elute the sample, prior to chromatography, would seem to be a prohibitive step for mass screening purposes. With some modifications, however, the "Berry" system might prove adaptable to mass screening. A circle could be punched from the urineimpregnated paper, and processed by amino acid chromatography in the manner described by Efron and colleagues. 1o This would provide a simple means to detect phosphoethanolaminuria in hypophosphatasia, cystathioninuria and argininosuccinic aciduria, all of which are ''high clearance" amino-acidopathies 28 and therefore not easily detected in plasma. The circles could also be processed by microbiological screening tests for "branched-chain" amino-aciduria,3 thereby providing additional assistance in detecting valinemia,32 maple syrup urine disease, Hartnup disease and many of the generalized amino-acidurias. 28 Sugar chromatography might also be applied to another circle of filter paper to identify mellituria and confirm galactosuria when suspected. 2. Guthrie and associates 13 , 14 have developed a simple method for the collection of whole blood on a special grade of filter paper, which
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is then analyzed by "inhibition assay" for abnormal concentrations of phenylalanine. The test is simple, sensitive and reliable. G, 25, 27 Yet in its present form the "inhibition assay" does not fulfill the criterion for "efficiency" in screening,19 since it will detect only a single disease. Guthrie 12 reports that microbiological methods and inhibition assays are now being developed which will provide multiple screening for alteration in the concentrations of leucine, isoleucine, valine, phenylalanine, histidine and galactose in a single specimen of blood. The "inhibition assay" has been used in a massive screening programme to detect phenylketonuria. H ,25 Undoubtedly this has had a profound impact on our society, not only through the increase in the incidence of early detection of phenylketonuria, but also in the stimulus it has given to planning and developing broader and more aggressive screening programmes.25 This endeavour has also had an immense effect on public awareness, and support for mass screening for metabolic disease. S. Two groups 10, 29 recently published descriptions of simple chromatographic techniques, applicable to plasma and urine samples, for detection of numerous amino-acidopathies. Chromatography of plasma is normally tedious if preliminary deproteinization is required. This obstacle was overcome when Culley and co-workers7 discovered that selection of the appropriate solvent allowed deproteinization and chromatography to be done directly on the filter paper. The two newer methods utilized this feature to process a sample of blood or plasma taken from infants, and by means of multiple stains in sequence used after chromatographic development could identify specifically most of the amino acids in plasma. Figure 57 gives some indication of the scope of these techniques. If urine is also screened in this manner, a great number of amino-acidopathies as well as disorders of carbohydrate metabolism can be identified. Confirmatory Testing In none of the present screening methods is a positive test result adequate evidence upon which to make a specific diagnosis. Confirmatory evidence is required from more specific analytic methods. Screening programmes must therefore include a depth of laboratory resources, to be called upon for confirmation or rejection of the tentative diagnosis raised by a positive screening test result. The requirement for high standards in screening, and the need for supplementary laboratory resources, suggest that large-scale screening should be done only in central laboratories. The Problem of Falsely Negative vs. Falsely Positive Results The ideal objective of any screening system will always be to identify all patients with disease. Because screening is based on an
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Figure 57. An example of multiple screening performed on single samples of plasma. Plasma (10 microliters) collected in microhematocrit tubes has been processed by a chromatographic method 29 and stained with a ninhydrin-isatin mixture (6 samples on left). Five known amino-acidopathies are illustrated in the samples obtained from patients with phenylketonuria (no. 2), hypermethioninemia (no. 3), tyrosinemia of prematurity (no. 4), citrullinemia (no. 5) and histidinemia (no. 6). Sample no. 1 is normal for comparison. Example no. 6' is the same as 6, but stained in sequence with the Pauly reagent for confirmation of histidine; example 5' is sample 5 stained in sequence with Ehrlich's reagent for confirmation of citrulline.
approximation, it is impossible to achieve this objective. A compromise must therefore be struck, the nature of which can be illustrated by the following example: The "inhibition assay" for detection of phenylketonuria will discriminate phenylalaninemia above 4 mg. per 100 ml.;13.14 the chromatographic methods 10. 29 cannot detect hyperphenylalaninemia with this sensitivity, nor with equal reliability. Since there will be some infants with phenylketonuria whose plasma phenylalanine levels may not be greatly elevated in the first week of life, 1 it would be desirable to use the more sensitive screening method. Alternatively, however, many normal infants can be expected to have temporarily elevated serum phenylalanine levels after birth.26 Therefore a greater number of falsely posi-
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tive results will be found if the more sensitive screening test is used for the detection of phenylketonuria. In other words, as one seeks to reduce the percentage of false negatives, by using a more sensitive method, there may be reciprocal increase in the percentage of falsely positive findings. This reciprocal trend has already been found in screening programmes with quite different intentions, using methods as different as cytology and roentgen examination. 15 Limits of confidence will have to be established for the meaningful interpretation of results obtained by screening tests; the price of accepting a certain percentage of false-negative results will have to be balanced against the burden of pursuing false-positive results. Current Outlook, and Problems in Mass Screening for Metabolic Disease Much has already been accomplished with mass screening tests for metabolic disease. Nevertheless there are many problems still to be resolved, and probably many more awaiting recognition. The pilot screening programmes now in existence are actually serving two purposes, fot while they are helping to increase the early detection of the diseases covered by the methods now in use, they are also highlighting the deficiencies in present-day approaches to mass screening." Legislation. The general interest in mass screening has stimulated new legislation in five states of the United States, and similar legislation is being proposed in several other states. The intent of this legislation is to ensure that a screening test for metabolic disease will be performed in early infancy on all babies. In some of these statutes there are specific recommendations concerning the age at which the test. must be done, the nature of the test and the disease to be sought. It seems advisable not to promote any legislation at present whose terms are too specific, and which may prove to be too restrictive, should future technical developments increase the scope of the screening programme. Furthermore, the whole problem of compulsory screening for diseases of very low frequency and no proved hazard to the general community is open to serious debate as to the moral and constitutional right of such legislation. Screening Directly for Modification of Proteins and Hormones in Body Fluids. The previous discussion on screening methods now in use implied limits to those methods. With few exceptions, they screen only for the intermediate compounds of cellular metabolism (e.g. amino acids, keto acids, monosaccharides). Mass screening for modification in the protein content of cells and body fluids has not been attempted, .. For example, MacCready reports (personal communication) that during the past 30 months the Massachusetts Department of Public Health has detected 27 patients with confirmed phenylketonuria, using the "inhibition assay";13 235,000 infants were tested. He also mentions that pilot studies are now in progress for the possible detection of other metabolic disease. Although the actual results of screening for phenylketonuria in this state are rewarding, it is the future results obtained in the expanded programme which will be even more instructive.
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nor has this been tried for the hormones. Further developments in this direction should be rewarding. 1. Studies in high-risk groups, and surveys have indicated that it is possible to screen directly and effectively for genetic variation in structure and activity of proteins (glucose-6-phosphate dehydrogenase, hemoglobin, transferrin, haptoglobin, albumin, ceruloplasmin, globulins, and so forth, have been "screened" in the past). Since the existence of these genetic variants (e.g. G-6-PD deficiency or pseudocholinesterase deficiency) may constitute a serious hazard to the subject, and since early recognition may modify the liability of the condition, there is justification in seeking methods for mass screening of these compounds. Development of automation of protein microelectrophoresis may be one source of progress in this area. Beutler and associates 5 have developed a test for galactosemia which may serve as a prototype for advances in screening for altered enzyme activity. The test is dependent on a colour change in the test solution, and it is sensitive enough to detect the heterozygote. The latter is a feature which ought to be of increasing importance in the future when identification of the heterozygote will be important in genetic counselling. 2. Mass screening for disturbances of hormonal content of body fluids has not been attempted. Since there are genetic or acquired disturbances which affect virtually all the endocrine organs and which can threaten health in the neonatal period, some rapid and simple indication of normal hormonal secretion rates would be of value. Screening of the Mother During Pregnancy. This is a well established practice for certain problems of metabolic significance to the offspring. The management of blood group incompatibility is one example. A broader scope to maternal screening is now being considered and will certainly become more important in the future, as increasing numbers of women, homozygous and heterozygous for metabolic diseases, bear children. 1. One new approach analyzes amniotic fluid for bilirubin concentration, in an effort to provide better management of hyperbilirubinemia. 22 As more is learned about the chemical composition of amniotic fluid during pregnancy, it may be possible to use this fluid to assist with antenatal assessment of other aspects of the infant's metabolism. But amniocentesis is not without hazard to mother and child,34 and it is therefore unlikely that antenatal screening of amniotic fluid will become a common procedure. 2. It may be important in coming years to have knowledge of the maternal genotype during pregnancy. Kang and co-workers17 have found that the mothers of phenylketonuric infants have a significant elevation of plasma phenylalanine concentration during pregnancy. Pregnancy therefore may facilitate detection of the heterozygote state.
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3. Approximately half of the patients with treatable hereditary metabolic disease who are detected and treated now as a result of mass screening will be homozygous but "healthy" women in the decades ahead. The majority of these women may want to bear children. During pregnancy the biochemical environment will be abnormal for the foetus if therapy for the disease is not continued. Preliminary evidence, obtained from studies in phenylketonuria,24 suggests that the intrauterine biochemical environment in the homozygous abnormal mother can damage the foetus. Maternal screening, or foreknowledge of the maternal phenotype, may therefore become as important as neonatal screening. This new challenge will be the legacy, perhaps unwanted, of today's progress in screening for metabolic disease. Screening for Chromosomal Abnormalities. The techniques for preparing reliable karyotypes are too complicated at present to be suitable for mass screening. But if, as suspected, 1 per cent of live births may be affected by chromosomal abnormality,23 then karyotype identification perhaps should be part of a screening programme. Chromosomal analysis is clearly of value for genetic counselling in those families in which trans locations are predisposing to autosomal nondisjunction. 23 In future, linkage studies for specific genes on autosomes may be accomplished if conjoint screening for metabolic and chromosomal abnormality is done. From such studies a preliminary linkage map is being assembled for the X chromosome, 9 and some advances in this direction are being attempted for autosomes. 21 Resources. Great demands will be made on the community undertaking mass screening, which will not be merely technical and organizational, pertaining to the screening procedures themselves. There would be little scientific or moral justification for mass screening for metabolic disease if it were undertaken without provision of ancillary resources. These important resources should include the following: (a) Laboratory services to provide continuing biochemical supervision of the patient's disease; (b) Hospital, clinic and consultative facilities, providing supervision for long-term care of patients with hereditary metabolic disease; (c) Provision of medicinal and dietary care, ensuring that the appropriate therapy is available commercially, and within the financial limits of the patient. (d) There is debate as to whether a registry of patients with heritable metabolic disease would be of value for genetic counselling and for tabulating the results and progress in mass screening. Ethical issues and considerations of the personal liberties of the citizen also temper discussion about the purpose of a registry for patients with heritable metabolic disease. (e) No comment has been made about the cost to the community for mass screening. The technical methods now in broad use
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are considered to be inexpensive. Nevertheless the costs for all aspects of large screening programmes will inevitably be great. Decisions apparently have already been made that mass screening can be worth while. A new challenge therefore faces the ingenuity and resourcefulness of our community-how best to support the costs of this new facet of medical progress.
CONCLUSION
Progress in medical care has brought mass screening for hereditary metabolic disease in the neonatal period to a position of prominence. Screening programmes of today are providing diagnostic services, as well as revealing deficits in our knowledge of this group of diseases. Because the techniques for screening and the designs of their application to the population are improving steadily, it is not advisable to designate any one technique as the "best" screening method at present. For this reason, . encouragement for mass screening should be liberal and foresighted. Careful review of the limits of the screening methods, and the limits to our knowledge of heritable metabolic disease, should direct our decisions on who should be screened, and when and how this is to be accomplished. There will be need for a complex structure within the community to meet all the needs of an adequate screening programme. In addition to the laboratory facilities and personnel required for the screening procedure itself, there should be supplementary and specific laboratory facilities to confirm diagnoses. There should also be facilities to ensure continuing medical care of the patient once identified. Assurance should be sought from industry that the complex modifications of environment which may be required to counteract the effects of genetic mutation will be available to the medical consumer.
APPENDIX
RECOMMENDATION OF THE COMMITTEE ON FETUS AND NEWBORN FOR THE SCREENING OF NEWBORN INFANTS FOR METABOLIC DISEASE 30
An opportunity to establish screening procedures for case-finding in a number of metabolic diseases now exists in the United States, because most infants are born in hospitals where appropriate screening can easily be carried out. Case finding in the neonatal period facilitates early inauguration of therapy, genetic counseling, and improved understanding of the natural history and incidence of metabolic diseases. The Committee on Fetus and Newborn considered four types of screening programs: (1) screening of all newborn infants, (2) screening of specific groups of neonates with increased risk of certain disorders, (3) large-scale, pilot-screening programs, designed primarily for research and acquisition of knowledge about the natural history
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of disease, (4) screening of expectant mothers, particularly using tests of amniotic fluid. The committee did not consider screening of older children. However, it is emphasized that a number of important diseases, e.g., Wilson's disease, cannot be detected by screening in the first few days of life. In evaluating screening tests for specific diseases, the Committee based its recommendations on the following criteria: 1. Does the seriousness of the disorder justify screening? 2. Is therapy for the disease in question available? 3. Is there a clearly identillable segment of the population with an increased incidence of this disease? 4. Is it possible to perform reliable screening during the first few days of life? 5. Can the screening test be performed in a routine service laboratory? 6. Is the test acceptable to the physician and to a majority of parents? 7. Is the cost of the test acceptable? 8. Are there acceptable medical facilities prepared to confirm diagnosis and consult about the institution of therapy?
RECOMMENDATIONS
At the present time the Committee believes the following recommendations on screening programs are iustilled:
Phenylketonuria A blood test for elevated concentration of phenylalanine performed no sooner than 24 hours after onset of milk feeding and prior to discharge is recommended for all newborn infants. " A second blood test at 4 or 6 weeks of age is recommended for all infants. This will detect infants who had borderline or low plasma concentrations of phenylalanine in the first few days of life. It will also confirm a positive initial test. Particular attention should be given to all newborn infants in families in which another member is already known to have phenylketonuria, with these infants tested daily during the hospital stay. If results are negative at discharge, the infant should be tested at 1, 2, and 6 weeks of age. Because of the difficulty of interpreting blood tests and the hazard of unwarranted dietary restrictions, it is recommended that the screening tests be performed in a large central facility, such as a state health department, or at least regional, laboratory. Only a very large facility will experience a sufficiently large number of tests positive for this rare disorder to acquire skill in diagnosis. It is also most important that the laboratory have a close working relationship with a medical center where the diagnosis can be confirmed, the treatment diet chemically monitored, and the therapy supervised.
Melituria Every newborn" should have a test for reducing substances (i.e., not utilizing glucose oxidase) in the urine on the day of discharge from the hospital. The test should be carried out by the individual hospital laboratory. It should be noted that metabolic disorders involving galactose and fructose (e.g., hereditary fructose intolerance) will not be detected in infants who have not been exposed to the substance in their diet, e.g., fructose excretion will appear only if sucrose or fructose were present in the feeding of an infant with fructose intolerance. For newborn siblings in families of known galactosemics the following test is recommended: heparinized cord blood should be obtained for measurement of galactose" Particular care must be exercised in interpreting results of tests in low birthweight infants for the accumulation of phenylalanine and the urinary excretion of reducing substances because positive findings may not indicate an inherited metabolic disOIder in this group of neonates.
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I-phosphate uridyl transferase activity. The infant should be placed on a lactose-free milk substitute until galactosemia can be ruled out. IT cord blood cannot be examined, a heparinized specimen of blood should be obtained as soon after birth as possible.
Other Metabolic Diseases Mter considering a number of other possible screening programs for diseases including maple syrup urine disease, fibrocystic disease, succinylcholinesterase deficiency, glucose 6-phosphatase dehydrogenase deficiency, cretinism, and gargoylism, the Committee believes that at the present time screening tests for these disorders are not ready for application to all newborns. All infants born into a family in which an inherited metabolic disorder has been recognized previously should be carefully evaluated in the neonatal period and appropriate screening tests should be performed wherever possible. The Committee urges that large-scale, research-oriented screening programs be undertaken at several centers; that several methods, including multiple inhibition assay, the newer chromatographic techniques developed for screening purposes, etc., be used in parallel, on blood samples; that all infants studied in the immediate neonatal period be studied again at 4-6 weeks. In this way incidence and natural history of a large number of inheritable metabolic diseases may be investigated. In addition, the most appropriate and efficient screening techniques can be determined. As knowledge accumulates such tests might become applicable to all newborns or older infants on a routine basis. . There is insufficient evidence at the present time to warrant screening of all expectant mothers.
REFERENCES 1. Armstrong, M. D.: Biochemistry; in F. L. Lyman (Ed.): Phenylketonuria. Springfield, Ill., Charles C Thomas, 1963, Chap. 4. 2. Berry, H. K.: Procedures for Testing Urine Specimens Dried on Filter Paper. Clin. Chem., 5:603, 1959. 3. Berry, H. K., Scheel, C., and Marks, J.: Procedures for Testing Urine Dried on Filter Paper: A Microbiological Test for Leucine, Valine and Isoleucine. Clin. Chern., 8:242, 1962. 4. Berry, H. K.: Detection of Metabolic Disorders among Mentally Retarded Children by Means of Paper Spot Tests. Am. I. Ment. Deficiency, 66:555, 1962. 5. Beutler, E., Baluda, M. C., and Donnell, G. N.: New Methods for Detection of Galactosemia and Its Carrier State. I. Lab. & Clin. Med., 64:694,1964. 6. Bixby, E. M., Pallatao, L. G., and pryles, C. V.: Evaluation of the Bacillus Subtilis Inhibition Assay Technique as a Screening Procedure for the Detection of Phenylketonuria. New England I. Med., 268:648, 1963. 7. Culley, W. J., Mertz, E. T., Luce, M. W., Calancro, J. M., and Jolty, D. H.: Paper Chromatographic Estimation of Phenylalanine and Tyrosine Using Finger Tip Blood. Clin. Chern., 8:266,1962. 8. Editorial: Screening for Galactosemia and Phenylketonuria. I.A.MA., 190:100, 1964. 9. Editorial: Genes on the X-chromosome. Canad. M.A.I., 89:222, 1963. 10. Efron, M. L., Young, D., Moser, H. W., and MacCready, R. A.: A Simple Chromatographic Screening Test for the Detection of Disorders of Amino Acid Metabolism. New England I. Med., 270: 1378, 1964. 11. Goldbloom, R. B., Sekeli, P., and Cameron, D.: Cystic Fibrosis of the Pancreas: Diagnosis by Application of a Sodium Electrode to the Skin. New England I. Med., 269:1349,1963. 12. Guthrie, R.: Routine Screening for Inborn Errors in the Newborn: "Instant Bacteria" Multiple Tests and "Inhibition Assays." Proc. Intemat. Congress on the Scientific Study of Mental Retardation. Copenhagen, August, 1964. 13. Guthrie, R., and Susi, A.: A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants. Pediatrics, 32:338, 1963.
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14. Guthrie, R., and \'Vhitney, S.: Phenylketonuria Detection in the Newborn Infant as a Routine Hospital Procedure. Children's Bureau Publication No. 419, 1964. U.S. Dept. of Health, Education, and Welfare. Welfare Admin., Washington, D.C. 15. Horvath, W. J.: The Effect of Physicians' Bias in Medical Diagnosis. Behavioural Science, 9:334, 1964. 16. Isselbacher, K. J.: Galactosemia; in J. B. Stanbury, J. B. Wyngaarden and D. S. Frederickson: The Metabolic BeMis of Inherited Disease. New York, McGrawHill Book Company, Inc., 1960, pp. 208-25. 17. Kang, E., Paine, R. S., Driscoll, K., and Wisniewski, B.: Elevation of Plasma Phenylalanine Levels during Pregnancies of Women Heterozygous for Phenylketonuria. ]. Pediat., 63:283, 1963. 18. Kleinman, D. S.: Phenylketonuria: A Review of Some Deficits in Our Information. Pediatrics, 33: 123, 1964. 19. Kleinman, D. S., and others: Phenylketonuria and the Guthrie Inhibition Assay Screening Procedure: Summary of Meeting of Consultants to the California State Department of Public Health. Pediatrics, 32:344, 1963. 20. Knox, W. E.: Phenylketonuria; in J. B. Stanbury, J. B. Wyngaarden and D. S. Frederickson: The Metabolic BeMis of Inherited Disease. New York, McGrawHill Book Company, Inc., 1960, pp. 321-2. 21. Lawler, S. D.: Localization of Autosomal Genes in Man. Human Bioi., 36: 146,1964. 22. Leading Article: Amniocentesis. Brit. M.j., 2:136, 1964. 23. Lejeune, J.: Autosomal Disorders. Pediatrics, 32:326, 1963. 24. Mabry, C. C., Denniston, J. C., Nelson, T. L., and Choon, D. S.: Maternal Phenylketonuria: A Cause of Mental Retardation in Children without the Metabolic Defect. New England]. Med., 269:1404, 1963. 25. MacCready, R. A., and Hussey, G. M.: Newborn Phenylketonuria Detection Project in Massachusetts. Am. ]. Pub. Health, 54:2075, 1964. 26. Menkes, J., and Avery, M. E.: The Metabolism of Phenylalanine and Tyrosine in the Premature Infant. Bull. Johns Hopkins Hosp., 113:301, 1963. 27. Partington, M. W., and Sinnott, B.: Case Finding in Phenylketonuria. II. The Guthrie Test. Ganad. M.A.j., 91:105, 1964. 28. Scriver, C. R.: Hereditary Aminoaciduria; in A. G. Steinberg and A. G. Beam (Eds.): Progress in Medical Genetics. New York, Grone & Stratton, Inc., 1962, pp. 83-186. 29. Scriver, C. R., Davies, E., and Cullen, A. M.: Application of a Simple Micromethod for the Screening of Plasma for a Variety of Aminoacidopathies. Lancet, 2:230, 1964. 30. Silverman, W. A., and others: American Academy of Pediatrics: Recommendation of the Committee on Fetus and Newborn for the Screening of Newborn Infants for Metabolic Diseases. Pediatrics, 35:499, 1965. 31. Somers, H. M., and Somers, A. R.: The Paradox of Medical Progress. New England J. Med., 266: 1253, 1962. 32. Wada, Y., and others: Idiopathic Valinemia. Tohoku J. Exper. Med., 81:46, 1963. 33. Wilson, J. M. G.: Multiple Screening. Lancet, 2:51, 1963. 34. Zipursky, A., Pollock, J., Chown, B., and Israels, L. G.: Transplacental Foetal Haemorrhage after Placental Injury during Delivery of Amniocentesis. Lancet, 2:493, 1963. Montreal Children's Hospital 2300 Tupper Street Montreal 25, P.Q. Canada
:\
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Many people regard mass screening for metabolic disease as an uninspired form of endeavour. One might therefore ask: Why is there a clinic in this volume devoted to the topic? One might also question why committees are convened, dissertations published, and mass screening programmes inaugurated, when the apparent objective is to find but one baby with a biochemical imbalance of hereditary or acquired origin, amongst the many thousands of normal infants. Advances in medical knowledge have brought contagion and undernutrition under control on our continent. The legacy of these advances has been a relative increase in the case load of the chronic, degenerative and heritable diseases. 31 Consequently the community has aligned its efforts, in order to bring these "new" problems under control. Since a heritable metabolic disease may produce its effect on the patient from the first day of life, it is the consensus that the earliest possible detection of such an abnormality is desirable. Mass screening in the neonatal period is one means by which this can be accomplished. The community, however, would not be interested in mass screening for metabolic diseases unless there were definite advantages to be accrued. There are three desirable results expected from mass screening:33 1. It provides, by means of an approximate and simple test, a method to segregate those who do have a disorder from those who do not. 2. Through early detection, appropriate therapy, when available, can be initiated before the disorder incapacitates the patient. 3. When the natural history of a specific disorder is poorly understood, mass screening can supply the data for better knowledge about the condition. Such programmes may have a fourth characteristic, particularly in reference to the study of rare, hereditary metabolic diseases. An ongoing screening project may provide new knowledge which will improve the screening procedure itself.
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It seems almost a matter of fact, therefore, that mass screening for metabolic disease should be instituted as soon as possible. Caution is nevertheless indicated, for there are many questions about screening, still requiring answers. Our knowledge of the exact role for mass screening in the control of metabolic disease is far from complete, even in such a well known condition as phenylketonuria. 18 The combined interest and efforts of pediatricians, obstetricians, public health authorities, biochemists and government representatives, as well as others, will be needed to solve many of the problems. The purpose of this clinic will be to examine, both in prospect and for the record, the role of mass screening in detection and study of the rare infant with genetically determined, biochemical imbalance. The problems and decisions in this field are in the hands of today's investigators. What is happening in this field now is anything but pedestrian, and because the community is so interested in what we are doing, there is unequaled opportunity to learn a great deal, and to do much that is good.
BIOLOGICAL PRINCIPLES UNDERLYING SCREENING FOR BIOCHEMICAL IMBALANCE OF HEREDITARY ORIGIN
Mass screening for metabolic disease relies on simple chemical tests, which must also be efficient, sensitive and reliable. For the moment, let us consider the nature of the biological problems being examined by these tests. Mutation will alter the information in the gene, and, subsequent to its translation, the initial cytoplasmic protein product will also be modified. Ultimately, the transformation and transduction of energy in the cell, dependent on that protein, may also be altered. The phenotype for the mutation may therefore be detectable as a modified protein geneproduct, or it may be identified only in the effect of this modification on cellular composition and activity. Screening methods may attempt to assess directly the protein constituents within cells (e.g. in red blood cells), or the proteins which are released from the cells into body fluids. Alternatively, the screening method may evaluate modification in the composition of the extracellular fluid, particularly plasma and urine. Figure 56 summarizes schematically the opportunities to detect the results of mutations affecting apoenzymes and other cellular proteins (site 1, Fig. 56), or affecting membrane transport processes (site 2, Fig. 56). Potential sources of the sample (A, B, C) for analysis are also indicated in the scheme. The analytical procedure which will identify the mutant phenotype must be simple for use in mass screening programmes; by similar token, collection of the biological sample to be tested must also be simple.
SCREENING NEWBORNS FOR HEREDITARY METABOLIC DISEASES
ECF,
S
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Plasma, etc. (8)
809
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~ pr0;lin
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E . . pI
(C) Urine
Figure 56. Schematic presentation of sites at which mutations might modify metabolism in a cell: 1, apoenzyme (E); 2, membrane transport (S -? S'). This might be identified by examining cytoplasmic proteins (A) themselves or the results of their modification of biochemical composition of plasma (B) and urine (C). S, Extracellular substrate; S', intracellular substrate; P', product formed from action of E and S'.
This, as we shall see, becomes an important consideration in screening of body fluids in the newborn infant. Blood and urine are the fluids most commonly used for mass screening of metabolic disease. Which one is chosen is dependent on precise knowledge of the kidney's role in biochemical homeostasis. Compounds with low renal clearance will be detected preferably in plasma. Compounds poorly transported by the tubules, from the glomerular filtrate, or secreted into the tubule lumen will be screened preferably in the urine. Heritable disturbances of membrane transport, phenotypically expressed in renal tubule cells, can be detected easily in urine, but only with poor reliability in plasma.
ATTITUDES AND PROBLEMS
Because there are so many problems pertaining to mass screening, a number of these will be discussed under separate headings. An overall perspective on current attitudes should be maintained, however, and the reports of two committees are recommended reading. One committee 19 analyzed the particular problems in screening for phenylketonuria; the other30 considered the broadest possible franchise for screening in the neonatal period (the text of the latter report is included in its entirety in the Appendix).
Who Should Be Screened? High-Risk Groups: A high-risk group comprises relatives of known patients with hereditary metabolic disease. Screening is most efficient
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when a proband has already been identified in a pedigree, and a search is made for other existing patients in that pedigree, or for new patients born into it. The methods used to find the high-risk patients are frequently chosen for their specificity in detecting the biochemical phenotype of the disease in question. The Population at Unpredicted Risk: This population offers the principal challenge to mass screening programmes. For this discussion the considerations are restricted to newborn infants, and in the present context this includes those infants still available for the first postnatal health examination. Obviously all newborn infants could be screened with considerable ease, because in North America most infants are born in hospital. This attitude is only partially acceptable, however, since it tends to exclude from mass screening programmes organized for hospital use those infants born at home. The problem is of significance for some of our rural communities, and particularly for other countries with obstetrical practices which are different from our own. What Diseases Should Be Screened? Screening is undoubtedly worth while for heritable metabolic disease with the following characteristics: (a) The natural history of the disease is well understood, and with few exceptions it can be reliably detected, early in life. (b) The disease, once identified by a screening procedure, can be confirmed by specific methods. (c) A therapeutic regimen is known which will alter the course of the untreated disease. (d) The therapeutic resources are easily available to the patient. Galactosemia 16 and phenylketonuria2o are examples of heritable metabolic disease with these attributes, which warrant mass screening for their early detection at the present time. 8 Alternatively, there are many diseases which do not fulfill all the criteria for mass screening. In that case, is there a reason not to screen for them? The following example illustrates this problem: The sodium content of sweat can be determined by a sodiumresponsive glass electrode applied to skin,u This method is easily applicable in the nursery, the determination requiring about three or four minutes for each infant. Data are presented in Table 36 which indicate that infants presenting with meconium ileus as a manifestation of fibrocystic disease can be distinguished from the normal infant in the first days of life. Whether all the infants who ultimately will be affected by fibrocystic disease can be discovered by this technique during the neonatal period will be determined only by further study. If it is found that infants with fibrocystic disease are detectable in the neonatal period, is one still justified in recommending mass screening of newborn infants for fibrocystic disease? Mass screening would be valid if there were an
811
SCREENING NEWBORNS FOR HEREDITARY METABOLIC DISEASES
Table 36.
Sodium Content oj Sweat in the Newborn (Glass Electrode Technigue: l1 mEg./L.)
DAY OF
NUMBER OF
LIFE
PATIENTS
1. ....... 100 2 ........ 100 3 ........ 99 4 ........ 100 5 ........ 87 6. . . . . . .. 10 12 ....... .
MEAN do S.D.
16.10 15.97 15.91 15.80 15.46 14.92
do do do do do do
5.82 3.97 4.17 4.17 4.04 3.04
ill
MECONIUM ILEUS PATIENTS
52.1 87.7 95.7 130.6
2
3
98.1 83.4
55.0
4
61.2 75.2 92.4
Unpublished data supplied by R. B. Goldbloom, P. Sekelj and D. Cameron from (1) 2511 testings taken from 5 sites (palms and dorsa of both hands, and forehead) in 100 healthy babies (58 male, 42 female) with mean birth Weight 3480 gm. (2) Four infants with proved meconium ileus.
unequivocal therapeutic advantage conferred on the fibrocystic infant, detected in the first week of life and treated accordingly, over the undetected, untreated fibrocystic infant. The data which will decide this issue are not yet available, because the role of early prophylactic therapy in fibrocystic disease has not been defined; the answers may be found through pilot projects using this new screening technique. When to Perform the Screening Test The answers to this question also depend on clear understanding of the phenotypic manifestations, and the course of the disease. In diseases which are detectable very early in life and which have serious consequences (e.g. galactosemia, phenylketonuria or maple syrup urine disease10 ) the earliest possible opportunity for screening is warranted, since delay in therapy usually results in devastation to the patient. In other cases there may be no apparent urgency for screening if the disease is benign; and again a disease whose phenotype becomes apparent only later in life cannot be detected in the neonatal period. Wilson's disease and cystinosis are examples of this type of metabolic disease. The natural course of the disease is not the only determinant of the optimum time for screening. The patterns of medical care for the newborn infant are also important; for example: 1. Tests for a modification of the biochemical composition of the body fluids must be performed when the imba:lance has become manifest. Dietary intake of the affected substance and postnatal age of the baby will both influence the degree of imbalance. 2. Very early discharge of the infant from the nursery may preclude reliable screening in hospital. The screening programme may have to be devised to reach the child at a later age. Screening may therefore be indicated on more than one occasion in the same infant. Present-day programmes usually operate during the
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first week of life at. the time the infant is discharged from the nursery, and again at the end of the first month of life.
METHODS AVAILABLE FOR LARGE·SCALE SCREENING PROGRAMMES
The analytical phase of the screening technique must be simple, efficient, sensitive and reliable. Whenever possible, it is preferable to use a method which will provide information on many diseases from a single biological sample. By comparison, a specific test for a single disease is uneconomical for screening populations in which there are many different hereditary diseases to be found. Collection of the sample must also be a simple procedure. Screening programmes for metabolic disease in older patients have often relied on urine collected in volume; but this can be a formidable procedure in the neonatal period or with an uncooperative patient. Several different approaches have been developed for collecting the sample and for identifying a mutant biochemical phenotype. 1. Berry2 has devised a simple method whereby urine is collected on a piece of filter paper inserted in the diaper. The dried urineimpregnated paper is then subjected to a variety of chemical spot tests. An aliquot of the urine may also be eluted from the paper and chromatographed in appropriate solvent systems. The advantages of this system include simple collection of the sample and a broad spectrum of tests. Any disease which will modify the composition of urine is potentially detectable by this method. The disadvantages are primarily technical. Soiling of the filter paper by feces may occur. The necessity to elute the sample, prior to chromatography, would seem to be a prohibitive step for mass screening purposes. With some modifications, however, the "Berry" system might prove adaptable to mass screening. A circle could be punched from the urineimpregnated paper, and processed by amino acid chromatography in the manner described by Efron and colleagues. 1o This would provide a simple means to detect phosphoethanolaminuria in hypophosphatasia, cystathioninuria and argininosuccinic aciduria, all of which are ''high clearance" amino-acidopathies 28 and therefore not easily detected in plasma. The circles could also be processed by microbiological screening tests for "branched-chain" amino-aciduria,3 thereby providing additional assistance in detecting valinemia,32 maple syrup urine disease, Hartnup disease and many of the generalized amino-acidurias. 28 Sugar chromatography might also be applied to another circle of filter paper to identify mellituria and confirm galactosuria when suspected. 2. Guthrie and associates 13 , 14 have developed a simple method for the collection of whole blood on a special grade of filter paper, which
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is then analyzed by "inhibition assay" for abnormal concentrations of phenylalanine. The test is simple, sensitive and reliable. G, 25, 27 Yet in its present form the "inhibition assay" does not fulfill the criterion for "efficiency" in screening,19 since it will detect only a single disease. Guthrie 12 reports that microbiological methods and inhibition assays are now being developed which will provide multiple screening for alteration in the concentrations of leucine, isoleucine, valine, phenylalanine, histidine and galactose in a single specimen of blood. The "inhibition assay" has been used in a massive screening programme to detect phenylketonuria. H ,25 Undoubtedly this has had a profound impact on our society, not only through the increase in the incidence of early detection of phenylketonuria, but also in the stimulus it has given to planning and developing broader and more aggressive screening programmes.25 This endeavour has also had an immense effect on public awareness, and support for mass screening for metabolic disease. S. Two groups 10, 29 recently published descriptions of simple chromatographic techniques, applicable to plasma and urine samples, for detection of numerous amino-acidopathies. Chromatography of plasma is normally tedious if preliminary deproteinization is required. This obstacle was overcome when Culley and co-workers7 discovered that selection of the appropriate solvent allowed deproteinization and chromatography to be done directly on the filter paper. The two newer methods utilized this feature to process a sample of blood or plasma taken from infants, and by means of multiple stains in sequence used after chromatographic development could identify specifically most of the amino acids in plasma. Figure 57 gives some indication of the scope of these techniques. If urine is also screened in this manner, a great number of amino-acidopathies as well as disorders of carbohydrate metabolism can be identified. Confirmatory Testing In none of the present screening methods is a positive test result adequate evidence upon which to make a specific diagnosis. Confirmatory evidence is required from more specific analytic methods. Screening programmes must therefore include a depth of laboratory resources, to be called upon for confirmation or rejection of the tentative diagnosis raised by a positive screening test result. The requirement for high standards in screening, and the need for supplementary laboratory resources, suggest that large-scale screening should be done only in central laboratories. The Problem of Falsely Negative vs. Falsely Positive Results The ideal objective of any screening system will always be to identify all patients with disease. Because screening is based on an
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Figure 57. An example of multiple screening performed on single samples of plasma. Plasma (10 microliters) collected in microhematocrit tubes has been processed by a chromatographic method 29 and stained with a ninhydrin-isatin mixture (6 samples on left). Five known amino-acidopathies are illustrated in the samples obtained from patients with phenylketonuria (no. 2), hypermethioninemia (no. 3), tyrosinemia of prematurity (no. 4), citrullinemia (no. 5) and histidinemia (no. 6). Sample no. 1 is normal for comparison. Example no. 6' is the same as 6, but stained in sequence with the Pauly reagent for confirmation of histidine; example 5' is sample 5 stained in sequence with Ehrlich's reagent for confirmation of citrulline.
approximation, it is impossible to achieve this objective. A compromise must therefore be struck, the nature of which can be illustrated by the following example: The "inhibition assay" for detection of phenylketonuria will discriminate phenylalaninemia above 4 mg. per 100 ml.;13.14 the chromatographic methods 10. 29 cannot detect hyperphenylalaninemia with this sensitivity, nor with equal reliability. Since there will be some infants with phenylketonuria whose plasma phenylalanine levels may not be greatly elevated in the first week of life, 1 it would be desirable to use the more sensitive screening method. Alternatively, however, many normal infants can be expected to have temporarily elevated serum phenylalanine levels after birth.26 Therefore a greater number of falsely posi-
SCREENING NEWBORNS FOR HEREDITARY METABOLIC DISEASES
815
tive results will be found if the more sensitive screening test is used for the detection of phenylketonuria. In other words, as one seeks to reduce the percentage of false negatives, by using a more sensitive method, there may be reciprocal increase in the percentage of falsely positive findings. This reciprocal trend has already been found in screening programmes with quite different intentions, using methods as different as cytology and roentgen examination. 15 Limits of confidence will have to be established for the meaningful interpretation of results obtained by screening tests; the price of accepting a certain percentage of false-negative results will have to be balanced against the burden of pursuing false-positive results. Current Outlook, and Problems in Mass Screening for Metabolic Disease Much has already been accomplished with mass screening tests for metabolic disease. Nevertheless there are many problems still to be resolved, and probably many more awaiting recognition. The pilot screening programmes now in existence are actually serving two purposes, fot while they are helping to increase the early detection of the diseases covered by the methods now in use, they are also highlighting the deficiencies in present-day approaches to mass screening." Legislation. The general interest in mass screening has stimulated new legislation in five states of the United States, and similar legislation is being proposed in several other states. The intent of this legislation is to ensure that a screening test for metabolic disease will be performed in early infancy on all babies. In some of these statutes there are specific recommendations concerning the age at which the test. must be done, the nature of the test and the disease to be sought. It seems advisable not to promote any legislation at present whose terms are too specific, and which may prove to be too restrictive, should future technical developments increase the scope of the screening programme. Furthermore, the whole problem of compulsory screening for diseases of very low frequency and no proved hazard to the general community is open to serious debate as to the moral and constitutional right of such legislation. Screening Directly for Modification of Proteins and Hormones in Body Fluids. The previous discussion on screening methods now in use implied limits to those methods. With few exceptions, they screen only for the intermediate compounds of cellular metabolism (e.g. amino acids, keto acids, monosaccharides). Mass screening for modification in the protein content of cells and body fluids has not been attempted, .. For example, MacCready reports (personal communication) that during the past 30 months the Massachusetts Department of Public Health has detected 27 patients with confirmed phenylketonuria, using the "inhibition assay";13 235,000 infants were tested. He also mentions that pilot studies are now in progress for the possible detection of other metabolic disease. Although the actual results of screening for phenylketonuria in this state are rewarding, it is the future results obtained in the expanded programme which will be even more instructive.
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nor has this been tried for the hormones. Further developments in this direction should be rewarding. 1. Studies in high-risk groups, and surveys have indicated that it is possible to screen directly and effectively for genetic variation in structure and activity of proteins (glucose-6-phosphate dehydrogenase, hemoglobin, transferrin, haptoglobin, albumin, ceruloplasmin, globulins, and so forth, have been "screened" in the past). Since the existence of these genetic variants (e.g. G-6-PD deficiency or pseudocholinesterase deficiency) may constitute a serious hazard to the subject, and since early recognition may modify the liability of the condition, there is justification in seeking methods for mass screening of these compounds. Development of automation of protein microelectrophoresis may be one source of progress in this area. Beutler and associates 5 have developed a test for galactosemia which may serve as a prototype for advances in screening for altered enzyme activity. The test is dependent on a colour change in the test solution, and it is sensitive enough to detect the heterozygote. The latter is a feature which ought to be of increasing importance in the future when identification of the heterozygote will be important in genetic counselling. 2. Mass screening for disturbances of hormonal content of body fluids has not been attempted. Since there are genetic or acquired disturbances which affect virtually all the endocrine organs and which can threaten health in the neonatal period, some rapid and simple indication of normal hormonal secretion rates would be of value. Screening of the Mother During Pregnancy. This is a well established practice for certain problems of metabolic significance to the offspring. The management of blood group incompatibility is one example. A broader scope to maternal screening is now being considered and will certainly become more important in the future, as increasing numbers of women, homozygous and heterozygous for metabolic diseases, bear children. 1. One new approach analyzes amniotic fluid for bilirubin concentration, in an effort to provide better management of hyperbilirubinemia. 22 As more is learned about the chemical composition of amniotic fluid during pregnancy, it may be possible to use this fluid to assist with antenatal assessment of other aspects of the infant's metabolism. But amniocentesis is not without hazard to mother and child,34 and it is therefore unlikely that antenatal screening of amniotic fluid will become a common procedure. 2. It may be important in coming years to have knowledge of the maternal genotype during pregnancy. Kang and co-workers17 have found that the mothers of phenylketonuric infants have a significant elevation of plasma phenylalanine concentration during pregnancy. Pregnancy therefore may facilitate detection of the heterozygote state.
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3. Approximately half of the patients with treatable hereditary metabolic disease who are detected and treated now as a result of mass screening will be homozygous but "healthy" women in the decades ahead. The majority of these women may want to bear children. During pregnancy the biochemical environment will be abnormal for the foetus if therapy for the disease is not continued. Preliminary evidence, obtained from studies in phenylketonuria,24 suggests that the intrauterine biochemical environment in the homozygous abnormal mother can damage the foetus. Maternal screening, or foreknowledge of the maternal phenotype, may therefore become as important as neonatal screening. This new challenge will be the legacy, perhaps unwanted, of today's progress in screening for metabolic disease. Screening for Chromosomal Abnormalities. The techniques for preparing reliable karyotypes are too complicated at present to be suitable for mass screening. But if, as suspected, 1 per cent of live births may be affected by chromosomal abnormality,23 then karyotype identification perhaps should be part of a screening programme. Chromosomal analysis is clearly of value for genetic counselling in those families in which trans locations are predisposing to autosomal nondisjunction. 23 In future, linkage studies for specific genes on autosomes may be accomplished if conjoint screening for metabolic and chromosomal abnormality is done. From such studies a preliminary linkage map is being assembled for the X chromosome, 9 and some advances in this direction are being attempted for autosomes. 21 Resources. Great demands will be made on the community undertaking mass screening, which will not be merely technical and organizational, pertaining to the screening procedures themselves. There would be little scientific or moral justification for mass screening for metabolic disease if it were undertaken without provision of ancillary resources. These important resources should include the following: (a) Laboratory services to provide continuing biochemical supervision of the patient's disease; (b) Hospital, clinic and consultative facilities, providing supervision for long-term care of patients with hereditary metabolic disease; (c) Provision of medicinal and dietary care, ensuring that the appropriate therapy is available commercially, and within the financial limits of the patient. (d) There is debate as to whether a registry of patients with heritable metabolic disease would be of value for genetic counselling and for tabulating the results and progress in mass screening. Ethical issues and considerations of the personal liberties of the citizen also temper discussion about the purpose of a registry for patients with heritable metabolic disease. (e) No comment has been made about the cost to the community for mass screening. The technical methods now in broad use
CHARLES R. SCRIVER
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are considered to be inexpensive. Nevertheless the costs for all aspects of large screening programmes will inevitably be great. Decisions apparently have already been made that mass screening can be worth while. A new challenge therefore faces the ingenuity and resourcefulness of our community-how best to support the costs of this new facet of medical progress.
CONCLUSION
Progress in medical care has brought mass screening for hereditary metabolic disease in the neonatal period to a position of prominence. Screening programmes of today are providing diagnostic services, as well as revealing deficits in our knowledge of this group of diseases. Because the techniques for screening and the designs of their application to the population are improving steadily, it is not advisable to designate any one technique as the "best" screening method at present. For this reason, . encouragement for mass screening should be liberal and foresighted. Careful review of the limits of the screening methods, and the limits to our knowledge of heritable metabolic disease, should direct our decisions on who should be screened, and when and how this is to be accomplished. There will be need for a complex structure within the community to meet all the needs of an adequate screening programme. In addition to the laboratory facilities and personnel required for the screening procedure itself, there should be supplementary and specific laboratory facilities to confirm diagnoses. There should also be facilities to ensure continuing medical care of the patient once identified. Assurance should be sought from industry that the complex modifications of environment which may be required to counteract the effects of genetic mutation will be available to the medical consumer.
APPENDIX
RECOMMENDATION OF THE COMMITTEE ON FETUS AND NEWBORN FOR THE SCREENING OF NEWBORN INFANTS FOR METABOLIC DISEASE 30
An opportunity to establish screening procedures for case-finding in a number of metabolic diseases now exists in the United States, because most infants are born in hospitals where appropriate screening can easily be carried out. Case finding in the neonatal period facilitates early inauguration of therapy, genetic counseling, and improved understanding of the natural history and incidence of metabolic diseases. The Committee on Fetus and Newborn considered four types of screening programs: (1) screening of all newborn infants, (2) screening of specific groups of neonates with increased risk of certain disorders, (3) large-scale, pilot-screening programs, designed primarily for research and acquisition of knowledge about the natural history
SCREENING NEWBORNS FOR HEREDITARY METABOLIC DISEASES
819
of disease, (4) screening of expectant mothers, particularly using tests of amniotic fluid. The committee did not consider screening of older children. However, it is emphasized that a number of important diseases, e.g., Wilson's disease, cannot be detected by screening in the first few days of life. In evaluating screening tests for specific diseases, the Committee based its recommendations on the following criteria: 1. Does the seriousness of the disorder justify screening? 2. Is therapy for the disease in question available? 3. Is there a clearly identillable segment of the population with an increased incidence of this disease? 4. Is it possible to perform reliable screening during the first few days of life? 5. Can the screening test be performed in a routine service laboratory? 6. Is the test acceptable to the physician and to a majority of parents? 7. Is the cost of the test acceptable? 8. Are there acceptable medical facilities prepared to confirm diagnosis and consult about the institution of therapy?
RECOMMENDATIONS
At the present time the Committee believes the following recommendations on screening programs are iustilled:
Phenylketonuria A blood test for elevated concentration of phenylalanine performed no sooner than 24 hours after onset of milk feeding and prior to discharge is recommended for all newborn infants. " A second blood test at 4 or 6 weeks of age is recommended for all infants. This will detect infants who had borderline or low plasma concentrations of phenylalanine in the first few days of life. It will also confirm a positive initial test. Particular attention should be given to all newborn infants in families in which another member is already known to have phenylketonuria, with these infants tested daily during the hospital stay. If results are negative at discharge, the infant should be tested at 1, 2, and 6 weeks of age. Because of the difficulty of interpreting blood tests and the hazard of unwarranted dietary restrictions, it is recommended that the screening tests be performed in a large central facility, such as a state health department, or at least regional, laboratory. Only a very large facility will experience a sufficiently large number of tests positive for this rare disorder to acquire skill in diagnosis. It is also most important that the laboratory have a close working relationship with a medical center where the diagnosis can be confirmed, the treatment diet chemically monitored, and the therapy supervised.
Melituria Every newborn" should have a test for reducing substances (i.e., not utilizing glucose oxidase) in the urine on the day of discharge from the hospital. The test should be carried out by the individual hospital laboratory. It should be noted that metabolic disorders involving galactose and fructose (e.g., hereditary fructose intolerance) will not be detected in infants who have not been exposed to the substance in their diet, e.g., fructose excretion will appear only if sucrose or fructose were present in the feeding of an infant with fructose intolerance. For newborn siblings in families of known galactosemics the following test is recommended: heparinized cord blood should be obtained for measurement of galactose" Particular care must be exercised in interpreting results of tests in low birthweight infants for the accumulation of phenylalanine and the urinary excretion of reducing substances because positive findings may not indicate an inherited metabolic disOIder in this group of neonates.
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I-phosphate uridyl transferase activity. The infant should be placed on a lactose-free milk substitute until galactosemia can be ruled out. IT cord blood cannot be examined, a heparinized specimen of blood should be obtained as soon after birth as possible.
Other Metabolic Diseases Mter considering a number of other possible screening programs for diseases including maple syrup urine disease, fibrocystic disease, succinylcholinesterase deficiency, glucose 6-phosphatase dehydrogenase deficiency, cretinism, and gargoylism, the Committee believes that at the present time screening tests for these disorders are not ready for application to all newborns. All infants born into a family in which an inherited metabolic disorder has been recognized previously should be carefully evaluated in the neonatal period and appropriate screening tests should be performed wherever possible. The Committee urges that large-scale, research-oriented screening programs be undertaken at several centers; that several methods, including multiple inhibition assay, the newer chromatographic techniques developed for screening purposes, etc., be used in parallel, on blood samples; that all infants studied in the immediate neonatal period be studied again at 4-6 weeks. In this way incidence and natural history of a large number of inheritable metabolic diseases may be investigated. In addition, the most appropriate and efficient screening techniques can be determined. As knowledge accumulates such tests might become applicable to all newborns or older infants on a routine basis. . There is insufficient evidence at the present time to warrant screening of all expectant mothers.
REFERENCES 1. Armstrong, M. D.: Biochemistry; in F. L. Lyman (Ed.): Phenylketonuria. Springfield, Ill., Charles C Thomas, 1963, Chap. 4. 2. Berry, H. K.: Procedures for Testing Urine Specimens Dried on Filter Paper. Clin. Chem., 5:603, 1959. 3. Berry, H. K., Scheel, C., and Marks, J.: Procedures for Testing Urine Dried on Filter Paper: A Microbiological Test for Leucine, Valine and Isoleucine. Clin. Chern., 8:242, 1962. 4. Berry, H. K.: Detection of Metabolic Disorders among Mentally Retarded Children by Means of Paper Spot Tests. Am. I. Ment. Deficiency, 66:555, 1962. 5. Beutler, E., Baluda, M. C., and Donnell, G. N.: New Methods for Detection of Galactosemia and Its Carrier State. I. Lab. & Clin. Med., 64:694,1964. 6. Bixby, E. M., Pallatao, L. G., and pryles, C. V.: Evaluation of the Bacillus Subtilis Inhibition Assay Technique as a Screening Procedure for the Detection of Phenylketonuria. New England I. Med., 268:648, 1963. 7. Culley, W. J., Mertz, E. T., Luce, M. W., Calancro, J. M., and Jolty, D. H.: Paper Chromatographic Estimation of Phenylalanine and Tyrosine Using Finger Tip Blood. Clin. Chern., 8:266,1962. 8. Editorial: Screening for Galactosemia and Phenylketonuria. I.A.MA., 190:100, 1964. 9. Editorial: Genes on the X-chromosome. Canad. M.A.I., 89:222, 1963. 10. Efron, M. L., Young, D., Moser, H. W., and MacCready, R. A.: A Simple Chromatographic Screening Test for the Detection of Disorders of Amino Acid Metabolism. New England I. Med., 270: 1378, 1964. 11. Goldbloom, R. B., Sekeli, P., and Cameron, D.: Cystic Fibrosis of the Pancreas: Diagnosis by Application of a Sodium Electrode to the Skin. New England I. Med., 269:1349,1963. 12. Guthrie, R.: Routine Screening for Inborn Errors in the Newborn: "Instant Bacteria" Multiple Tests and "Inhibition Assays." Proc. Intemat. Congress on the Scientific Study of Mental Retardation. Copenhagen, August, 1964. 13. Guthrie, R., and Susi, A.: A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants. Pediatrics, 32:338, 1963.
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14. Guthrie, R., and \'Vhitney, S.: Phenylketonuria Detection in the Newborn Infant as a Routine Hospital Procedure. Children's Bureau Publication No. 419, 1964. U.S. Dept. of Health, Education, and Welfare. Welfare Admin., Washington, D.C. 15. Horvath, W. J.: The Effect of Physicians' Bias in Medical Diagnosis. Behavioural Science, 9:334, 1964. 16. Isselbacher, K. J.: Galactosemia; in J. B. Stanbury, J. B. Wyngaarden and D. S. Frederickson: The Metabolic BeMis of Inherited Disease. New York, McGrawHill Book Company, Inc., 1960, pp. 208-25. 17. Kang, E., Paine, R. S., Driscoll, K., and Wisniewski, B.: Elevation of Plasma Phenylalanine Levels during Pregnancies of Women Heterozygous for Phenylketonuria. ]. Pediat., 63:283, 1963. 18. Kleinman, D. S.: Phenylketonuria: A Review of Some Deficits in Our Information. Pediatrics, 33: 123, 1964. 19. Kleinman, D. S., and others: Phenylketonuria and the Guthrie Inhibition Assay Screening Procedure: Summary of Meeting of Consultants to the California State Department of Public Health. Pediatrics, 32:344, 1963. 20. Knox, W. E.: Phenylketonuria; in J. B. Stanbury, J. B. Wyngaarden and D. S. Frederickson: The Metabolic BeMis of Inherited Disease. New York, McGrawHill Book Company, Inc., 1960, pp. 321-2. 21. Lawler, S. D.: Localization of Autosomal Genes in Man. Human Bioi., 36: 146,1964. 22. Leading Article: Amniocentesis. Brit. M.j., 2:136, 1964. 23. Lejeune, J.: Autosomal Disorders. Pediatrics, 32:326, 1963. 24. Mabry, C. C., Denniston, J. C., Nelson, T. L., and Choon, D. S.: Maternal Phenylketonuria: A Cause of Mental Retardation in Children without the Metabolic Defect. New England]. Med., 269:1404, 1963. 25. MacCready, R. A., and Hussey, G. M.: Newborn Phenylketonuria Detection Project in Massachusetts. Am. ]. Pub. Health, 54:2075, 1964. 26. Menkes, J., and Avery, M. E.: The Metabolism of Phenylalanine and Tyrosine in the Premature Infant. Bull. Johns Hopkins Hosp., 113:301, 1963. 27. Partington, M. W., and Sinnott, B.: Case Finding in Phenylketonuria. II. The Guthrie Test. Ganad. M.A.j., 91:105, 1964. 28. Scriver, C. R.: Hereditary Aminoaciduria; in A. G. Steinberg and A. G. Beam (Eds.): Progress in Medical Genetics. New York, Grone & Stratton, Inc., 1962, pp. 83-186. 29. Scriver, C. R., Davies, E., and Cullen, A. M.: Application of a Simple Micromethod for the Screening of Plasma for a Variety of Aminoacidopathies. Lancet, 2:230, 1964. 30. Silverman, W. A., and others: American Academy of Pediatrics: Recommendation of the Committee on Fetus and Newborn for the Screening of Newborn Infants for Metabolic Diseases. Pediatrics, 35:499, 1965. 31. Somers, H. M., and Somers, A. R.: The Paradox of Medical Progress. New England J. Med., 266: 1253, 1962. 32. Wada, Y., and others: Idiopathic Valinemia. Tohoku J. Exper. Med., 81:46, 1963. 33. Wilson, J. M. G.: Multiple Screening. Lancet, 2:51, 1963. 34. Zipursky, A., Pollock, J., Chown, B., and Israels, L. G.: Transplacental Foetal Haemorrhage after Placental Injury during Delivery of Amniocentesis. Lancet, 2:493, 1963. Montreal Children's Hospital 2300 Tupper Street Montreal 25, P.Q. Canada
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