10 Neonatal screening for inborn errors of amino acid metabolism

10 Neonatal Screening for Inborn Errors of Amino Acid Metabolism HARVEY

L. L E V Y

Though neonatal screening for inborn errors of amino acid metabolism did not begin in any substantial way until the early 1960s, such screening was made inevitable several years before by one of the most important discoveries in this field. This discovery, by Professor Horst Bickel, the editor of this issue, was that phenylketonuria (PKU), a disease known to cause brain damage with mental retardation, could be controlled biochemically and to some extent clinically by a special diet containing only relatively small amounts of the naturally-occurring and essential amino acid phenylalanine (Bickel, Gerrard and Hickmanns, 1953). In subsequent years it was shown that such a diet when begun in the neonatal period and continued at least through early childhood could even prevent altogether the neurological damage of P K U (Knox, 1960). So when Professor Guthrie of Buffalo in 1961 introduced his brilliantly simple and inexpensive test for the general population screening of PKU in neonates, he could realise that it would be an invaluable tool whereby such neonates would be discovered in time for dietary therapy to be effective (Guthrie, 1961). The discovery of the 'Guthrie' test is a rather interesting story. In the 1950s Guthrie had developed certain bacterial assays as a method of detecting biochemical metabolites in human specimens. These methods were used to measure the effects of chemotherapy in cancer. However, in the late 1950s he was asked to monitor the blood phenylalanine concentrations of two patients with PKU. Befitting his scientific acumen, he shortly succeeded in modifying his assay so that it was specifically quantitative for phenylalanine as the free amino acid (Guthrie, 1972). Using it to test blood specimens from mental retardates in a New York institution, he showed that those with P K U could very easily be distinguished biochemically on the basis of the response to this test. By 1961 Professor Guthrie had developed this assay so that it could be easily performed on dried blood that had been impregnated into filter paper. This assay was then used in Massachusetts by Dr Robert MacCready, Director of the Diagnostic Laboratories for the Massachusetts Department of Public Health, who, with his quiet perseverance and selfless devotion to the Clinics in Endocrinology and Metabolism--Vol. 3, No. 1, March 1974.

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prevention of disease, shortly succeeded in convincing both hospital pathologists and paediatricians to submit a filter-paper blood specimen on each newborn before nursery discharge. This was the first general newborn blood screening program for any inborn error of metabolism (MacCready and Hussey, 1964). Within a few years similar neonatal screening programs for PKU were underway in many other states within the United States and in other areas of the world. In addition, this led to a general concept of neonatal screening that now includes not only inborn errors of amino acid metabolism other than PKU but other genetic disorders as well (Levy, 1974). TESTS FOR A M I N O ACID SCREENING Bacterial assays

The assays used for screening are so closely allied to the programs themselves and to the resulting clinical considerations that it is worth at least briefly discussing them. The mainstay of such screening at present is the 'Guthrie' test mentioned above. This is a bacterial inhibition assay. The principle is that bacterial growth, which would normally occur in a proper medium, is inhibited by an analogue (e.g. B-2 thienylalanine) of the amino acid in question (e.g. phenylalanine). However, the analogue can itself be counteracted by the amino acid (presumably a competitive situation) so that growth occurs in the presence of the specific amino acid (Figure 1). An important

BACTERIA

+

MEDIUM

BACTERIA

+

MEDIUM

~'k~"~c"~c" BACTERIA +

MEDIUM

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% Figure I. Diagrammatic representation of the mechanism of the "Guthrie" bacterial inhibition assay. In this depiction it is suggested that the amino acid competes with the analogue at the level of cellular transport. This may be true though it is equally plausible that the competition is at the intracellular metabolic level. aspect of this assay is that when the concentration of the inhibiting analogue is kept constant, the amount of bacterial growth is directly proportional to the concentration of the amino acid. In practice, the assay is performed in plastic trays containing agar, a medium consisting of various inorganic and organic compounds, bacteria (Bacillus subtilis), and a specific inhibitor (analogue). After these materials have been pipetted into the trays and the agar

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solidified, a disc from each filter-paper blood specimen is punched by hand or by a special punch index machine (Fundamental Products, Inc., North Hollywood, California), onto the agar. Each tray contains, in addition to the newborn blood discs, a row of control blood discs containing varying amounts of the amino acid. After complete preparation, the trays are incubated at 37°C overnight, following which the growth around each disc is observed (Guthrie and Susi, 1963). As noted in Figure 2, a disc of blood containing a large concentration of an amino acid is readily distinguished from those discs with a normal amino acid concentration.

Figure 2. A typical "Guthrie' plate for phenylalanine. The centre row contains dried horseblood filter-paper discs used as controls with concentrations of phenylalanine varying from less than 2 mg/100 ml to 20 mg/100 ml. In the second row from the top is a blood specimen from a neonate indicating marked hyperphenylaIaninaemia (phenylalanine concentration almost 20 mg/100 ml). This infant was later proved to have phenylketonuria. As mentioned above, the first ~Guthrie' bacterial inhibition assay developed was for phenylalanine and served to identify infants with PKU. However, Guthrie and his group soon modified the original test so that other amino acid accumulations could be detected. The modification mainly involved changing the inhibitor to an analogue of the amino acid in question. In some instances the strain of B. subtilis also had to be changed. Today there are five bacterial inhibition assays available for the purpose of neonatal screening. |n Table 1 are listed these assays, the inhibitor used, and the specific disorder sought.

Chromatography Soon after general neonatal screening was made possible by the ~Guthrie' tests, two paper chromatographic methods for screening were almost simultaneously introduced. It is significant that each method was developed by a

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former student of Professor Charles Dent of London, who several years before was the first to simplify paper chromatography for amino acid analysis and to apply this technique to the discovery and investigation of amino acid disorders (Dent, 1946). The 'Efron' method, developed by Dr Mary Efron of Boston, utilises either the filter-paper whole blood specimens used in the 'Guthrie' test or filter-paper urine specimens (Efron et al, 1964). This method simply consists of placing discs of these specimens into pre-punched holes in chromatography paper, allowing chromatography to proceed overnight in a tank containing a suitable solvent (usually butanol-acetic acid-water, 12:3:5), and then staining the dried chromatograms with ninhydrin so as to visualise the amino acids (Figure 3). The ~Scriver' method, developed by

Figure 3. An amino acid chromatogram performed by the 'Efron' technique of paper chromatography. In the centre (AA) is a disc containing reference standards of free amino acids. Near this disc (15) is a dried-blood filter-paper disc of an infant with hypermethioninaemia. All other discs represent the normal whole blood amino acid pattern.

Professor Charles Scriver of Montreal, utilises liquid serum instead of dried whole blood (Scriver, Davies and Cullen, 1964). The serum is applied to chromatography paper and chromatography and subsequent staining proceed as in the 'Efron' method above. In either method, as in the more recently developed methods of chromatography by thin-layer techniques (Nfitzenadel and Lutz, 1970), abnormalities are detected by 'spots' of increased intensity or by 'spots' in unusual locations (Figure 3),

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Bacterial assay vs. chromatography

For several years considerable controversy has raged over the relative merits and liabilities of these two forms of screening for the amino acid disorders. On the one hand, chromatography offers the opportunity to visualise all amino acids in a specimen by a single test. Thus several different disorders could theoretically be detected at one time. By contrast, only one amino acid and thus usually only one disorder can be detected in each bacterial assay. However, the bacterial inhibition assays are more sensitive than is chromatography and thus there is less chance of 'missing' a disorder by bacterial inhibition assay than there is by chromatography. Furthermore, since the materials for bacterial inhibition assays are less expensive than those necessary for chromatography, it is usually more efficient to perform bacterial inhibition assays in a large laboratory. In summary, it would seem that a comprehensive neonatal screening program for amino acid disorders could best utilise bacterial inhibition asasys for those disorders detectable in this manner and some form of chromatography (paper or thin-layer) for other amino acid disorders.

CURRENT SCREENING PROGRAMS Blood

PKU is currently the most common and most important disease sought by screening the blood of newborns. In 45 of the 50 states within the United States and in most of the European countries as well as a few countries in Asia, the Middle East, and the South Pacific, screening for P K U among newborns is conducted to at least some degree. In the United States the screening facility is usually the central public health laboratory of the state, though in a few states, notably California, screening is conducted in many relatively small private hospitals. [n Europe and elsewhere, screening is usually conducted in one or more large central university hospitals. The screening procedure is similar in all areas. On the day the newborn is to be discharged from the nursery, a blood specimen is obtained from the heel by capillary puncture. If the 'Guthrie' test or the 'Efron' method of paper chromatography is to be the method of analysis, the blood specimen is directly impregnated into a filter-paper card (Figure 4) and, when dry, mailed to the central laboratory. If the 'Scriver' method of paper chromatography is the testing method, the blood is taken up into capillary tubes and mailed to the central laboratory. At the laboratory performing the 'Guthrie' test, specimens are sorted, the trays are sterilised, a 1/8-in disc from each specimen is placed on a tray, the trays are incubated overnight, and the following morning the growth zones surrounding each disc on a tray are observed. Increased growth around a disc, as noted in Figure 2, may indicate the presence of PKU. If paper chromatography is the method of analysis, a 3/16-in disc (equivalent to 10 microlitres of whole blood) from each filter paper specimen ('Efron method') or 5 microlitres of serum ('Scriver' method) are placed at the lower end of the chromatography paper and the procedure continues as previously described.

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Figure 4. Impregnation of blood from the heel of a newborn into a filter-paper card. In a few areas, notably the states of Massachusetts, Oregon and New York, in New Zealand and in the Heidelberg, Germany and Vienna, Austria districts, disorders in addition to P K U are specifically sought in newborns. For this purpose, multiple 'Guthrie' bacterial inhibition assays, as listed in Table 1, are used (Guthrie and Murphey, 1971). The procedure is identical to that described above except that discs from each dried-blood filter-paper specimens are placed on several different trays rather than on only the single tray for phenylalanine. Of course, those areas that screen blood by chromatographic methods, including several Canadian provinces, as well as Manchester and Liverpool, England, would also identify disorders other than PKU. Table 1. 'Guthrie' bacterial inhibition assays currently in use ['or neonatal screening of the inborn errors o f amino acM metabolism Amino acid

Inhibitor

Disorder

Phenylalanine Leucine

DL-~-2-thienylalanine 4-aza-leucine HCI

Methionine

DL-methionine DLsulphoximine D-tyrosine 1,2,4-triazole-3-alanine

Phenylketonuria (PKU) Maple syrup urine disease (MSUD) Homocystinuria

Tyrosine Histidine

Tyrosinaemia Histidinaemia

Urine The emphasis in neonatal screening has been on blood testing. This is understandable since the primary disorder sought has been P K U and this is much more readily identified in blood than in other body fluids of the neonate. However, a number of amino acid disorders are not identifiable in blood by

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present newborn screening methods and a few cannot possibly be identified in blood under any circumstances since no known blood abnormality exists. Among the former are such metabolic disorders as argininosuceinic acidaemia, hyperlysinaemia, hyperglycinaemia, propionic acidaemia, methylmalonic acidaemia, and isovaleric acidaemia and among the latter are such transport disorders as cystinuria, Hartnup 'disease', iminoglycinuria and the Fanconi syndrome. It is now apparent, however, that these disorders as well as many others can be identified by relatively simple urine screening methods. Two areas of the world, Massachusetts and the state of New South Wales in Australia, have pioneered in this form of neonatal screening (Levy, Shih and Madigan, 1972; Turner and Brown, 1972). In Massachusetts a filterpaper specimen of urine is obtained by the parent at home when the infant

Figure 5. Paper chromatography of urine performed by the 'Efron' method. The abnormal amino acid patterns are readily distinguishable from the normal (N) patterns. The following disorders are represented (from left to right): Hartnup 'disease' (H); cystinuria (C); hyperglyeinaemia (G). DC represents an artifactual pattern resembling the increased branched-chain amino acid excretion of maple syrup urine disease but due to contamination of the filter paper by a proprietary diaper cream. A reference standard of free amino acids is in the centre.

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is three to four weeks old and mailed to the central state laboratory. Here the specimen is tested by the 'Efron' method of paper chromatography. Disorders identifiable in urine are usually readily distinguishable by a distinctly abnormal amino acid pattern (Figure 5). In the state of New South Wales in Australia, neonatal urine dried into filter paper is obtained in a similar manner but the paper chromatographic method of analysis is that of Berry (1959), in which the urine is first eluted from the filter paper and then 'spotted' on the chromatography paper.

Comprehensive screening The most comprehensive neonatal screening program for amino acid disorders currently being conducted is in Massachusetts. This program is known as the Massachusetts Metabolic Disorders Screening Program. Three separate specimens are solicited and generally obtained on each infant. As listed in Table 2, a filter-paper umbilical cord blood specimen is obtained at birth followed by a filter-paper capillary blood specimen on the third to fifth day of life (usually at nursery discharge) and finally a filter-paper urine specimen obtained by the parent at home when the infant is three to four weeks of age. As will be discussed later, a/most all of the disorders listed in Table 2 plus additional disorders have been detected by this composite screening.

Table 2. Specimens and tests performed on newborns in the Massachusetts Metabolic Disorders Screening Program

Specimena

Age

Tests

Disorder(s)

Cord blood

Birth

Capillary blood

3-5 d a y s

'Classical' galactosaemia Maternal phenylketonuria Phenylketonuria Maple syrup urine disease Homocystinuria Tyrosinosis Galactosaemias

Urine

3-4 weeks

'Beutler' enzyme assay GBIA (Phe)b GBIA (Phe) GIBA (Leu) GBIA (Meth) GBIA (Tyr) ~Paigen' galactose assay Paperchromatography

Many other amino acid disorders

'All specimens are impregnated into filter paper. O'Gutbrie' bacterial inhibition assays; Phe (phenylalanine), Leu (leucine), Meth (metbionine), Tyr (tyrosine).

RESULTS OF N E O N A T A L S C R E E N I N G

Neonatal frequency of disorders In a comprehensive program about one infant in every 2500 screened will have a definable primary disorder of amino acid metabolism or transport. Thus in Massachusetts approximately 30 to 35 infants are identified each year among the 75 000 to 85 000 screened. Additional infants with amino acid abnormalities secondary to other disorders are also identified. For instance,

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increases in the blood c o n c e n t r a t i o n s o f m e t h i o n i n e and tyrosine are frequently detected in association with neonatal hepatocellular disease and m a y even be the first indication o f such disease. F u r t h e r m o r e , one infant in M a s s a c h u setts with generalised h y p e r a m i n o a c i d u r i a was d e t e r m i n e d upon further investigation to have vitamin D - d e p e n d e n t rickets. Table 3 lists the a p p r o x i m a t e n u m b e r o f neonates discovered to have certain a m i n o acid disorders and the frequency o f such disorders in the n e w b o r n p o p u l a t i o n , as based on w o r l d w i d e results o f screening. However, these d a t a are by no means definitive. The criteria for a specific diagnostic classification and the completeness o f follow-up m a y differ rather m a r k e d l y f r o m one a r e a to another, thus significantly affecting the r e p o r t e d frequency. F u r t h e r m o r e , it is k n o w n that there m a y be rather wide genetic variations a m o n g different p o p u l a t i o n s ( T h a l h a m m e r a n d Scheiber, 1972), thereby raising the possibility t h a t d a t a f r o m n e w b o r n p o p u l a t i o n s not now being screened could m a r k e d l y alter these reported frequencies.

Table 3. Worldwide results o f routine newborn screening .('or inborn errors o f amino acid metabolism a

Disorder Phenylketonuria b Maple syrup urine disease Homocystinuria Tyrosinosis Histidinaemia Hartnup 'disease' Cystinuria Iminoglycinuria Hyperprolinaemia Cystathioninaemia Argininosuccinic acidaemia Hyperglycinaemia (nonketotic) Hyperlysinaemia Propionic acidaemia Fanconi syndrome

Number screened

Number identified

Newborn frequency

13 665 644 3 372 363 2 781 042 1 528 509 1 098 215 651 323 651 323 651 323 631 025 564 025 364 025

1190 17 12 0 47 26 83 53 4 5 4

1:11 500 1:200 000 1:230 000 1:23 000 1:25 000 1:8000 1:12 000 1 : 150 000 1:110 000 1:90 000

364 025 364 025 364 025 364 025

2 1 1 1

l:180 000 < 1:300 000 < 1:300 000 < 1:300 00q

aMost of these data were compiled at the end of 1971 but more recent data from Massachusetts have been included. bOnly infants considered to have 'classical' PKU are included in this table.

In Massachusetts careful investigations have been c o n d u c t e d for several years on infants a n d their families identified by n e w b o r n screening. On the basis o f these studies, it is now possible to place each detected a m i n o acid d i s o r d e r into one o f several frequency groups, as listed in Table 4. As can be j u d g e d f r o m c o m p a r i n g this table to Table 2, the m o s t frequent disorders are those detected by urine screening. However, as discussed below, m a n y o f the m o s t clinically significant disorders are detected by the screening o f blood.

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H.L. LEVY Table 4. Classification o f amino acM disorders as based on neonatal frequency in Massachusetts Group I (1:10 000-1:20 000)

Phenylketonuria (PKU) Hyperphenylalaninaemia (non-PKU) Histidinaemia Iminoglycinuria Cystinuria Hartnup disorder Group 11 (< 1:20 000-1:100 000) Argininosuccinic acidaemia Group 111 (< 1:100 000-1:200 000)

Cystathioninaemia Homocystinuria Hyperglycinaemia (non-ketotic) Maple syrup urine disease Group I V (< 1:300 000) Fanconi syndrome Hyperlysinaemia Hyperornithinaemia Propionic acidaemia Hyperprolinaemia

Clinical significance Certainly the most important consideration in neonatal screening is the effect that the untreated disorder may have on the individual. Early studies on a few affected individuals identified as a result of clinical disease suggested that virtually all amino acid disorders were serious clinical entities (Ghadimi, 1967). Such is certainly not the case. One of the most important results of routine neonatal screening has been to demonstrate that some of these disorders are benign. Though the investigation is still in its early stages, the Massachusetts studies strongly suggest that about 60 per cent of the disorders identified in this newborn screening program are clinically significant and the remaining 40 per cent are probably benign (Figure 6). Furthermore, it Table 5. Amino acM disorders believed to be clinically significant and those believed to be benign, as based on studies in Massachusetts

Immediate significance

Delayed significance Benign

Phenylketonuria (PKU) Hyperphenylalaninaemia ('high variant') Maple syrup urine disease Homocystinuria (certain types) Argininosuccinic acidaemia (certain types) Hyperglycinaemia, non-ketotic Propionic acidaemia

Cystinuria Fanconi syndrome

Hyperphenylalaninaemia (mild) Histidinaemia Cystathioninaemia Iminoglycinuria Hartnup 'disease'

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N E O N A T A L S C R E E N I N G F O R I N B O R N ERRORS OF A M I N O A C I D METABOLISM

appears that about 50 per cent of all infants detected have a clinically insignificant disorder (Figure 7). Table 5 lists the disorders believed to have significance in terms of medical immediacy as opposed to the disorders which seem to have significance later in life and the other disorders which appear to be benign. In comparing this table to Table 4, it is apparent that several disorders in the highest frequency category are seemingly benign, though phenylketonuria is a striking exception.

Figure 6. Percentage of clinically significant disorders among all amino acid disorders detected by the Massachusetts program.

/ .,j j - ' /

From this discussion it might seem that neonatal screening for amino acid disorders other than phenylketonuria is not worthwhile. Such is not the case. It is now apparent that maple syrup urine disease, including its less severe variants, is a serious disorder and that treatment beginning early in the neonatal period may be the only present means of preventing the clinical complications. Similarly, homocystinuria due to cystathionine synthase deficiency may be devastating in its clinical effects. The same may be said for

Figure 7. Percentages of infants with disorders of immediate significance (40 per cent) and of infants with disorders of delayed significance (10 per cent) among all infants with amino acid disorders detected by the Massachusetts program.

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i //

\\ \

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a number of other disorders, including argininosuccinic acidaemia, nonketotic hyperglycinaemia, propionic acidaemia, and so forth. Furthermore, refinement of our current screening techniques will almost surely result in the detection of many additional infants with these serious disorders who may now be overlooked or even the detection of infants with serious but yet undiscovered amino acid disorders.

164

H.L. LEVY PITFALLS OF SCREENING

Screening vs. diagnosis One of the most important concepts to appreciate in regard to screening is that any good neonatal screening procedure merely eliminates most infants from consideration as having particular disorders and leads to suspicion in a few infants. By no means should suspicion be equated with diagnosis. The vast majority of neonates with mild increases in the blood phenylalanine concentration will not have PKU though all should be retested in order that the occasional infant with PKU can be selected and subsequently diagnosed. Similarly, increases in the blood methionine concentration (Levy et al, 1969b) or in the urinary excretion of certain amino acids (Levy et al~ 1972) may be noted in early infancy either as transient findings of no clinical consequence or as due to a specific metabolic disorder. The normality of such findings as well as the presence of a disorder can only be established by careful follow-up with repeat testing in all such infants and by specific confirmatory testing when indicated by the persistence or magnitude of the findings. Artifacts In any large testing program artifacts can present significant problems. Early in the Massachusetts program it was discovered that an appearance of hyperaminoaciduria could be produced by inadvertent faecal contamination of the newborn urine specimen submitted for screening (Levy, Madigan and Lum, 1969a). Subsequently, it became apparent that certain infant formulas fortified with DL-methionine could produce marked methioninuria (Efron et al, 1969), that certain diaper creams could produce an amino acid pattern on a filter paper urine specimen suggesting maple syrup urine disease (Levy et al, 1972; Figure 5) and that squamae on the filter-paper blood specimen could produce a 'positive' result on a test for maple syrup urine disease (Burns and Vanderlinde, 1971). As in transient findings, only by careful follow-up and inspection of the original specimens will such artifacts be distinguished from an inborn error of amino acid metabolism.

SUMMARY Neonatal screening for the inborn errors of amino acid metabolism and transport, begun as only phenylketonuria (PKU) screening, is gradually becoming more widespread and encompassing many different disorders. The basic screening methods used are the.'Guthrie' tests and, in some instances, paper chromatography. P K U still ranks as the most important disorder sought, in terms of frequency and effectiveness of treatment. However, other disorders, such as maple syrup urine disease and homocystinuria, are also important. Neonatal screening, in addition to identifying affected infants for the purpose of therapy, will also result in a vast amount of new information of immeasurable importance to our understanding of the role of these disorders in clinical disease. However, before therapy can be instituted

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or accurate i n f o r m a t i o n gained, it is imperative that p r o p e r diagnosis be m a d e by the use o f repeat testing and c o n f i r m a t o r y procedures. Artifacts and transient findings often m i m i c the findings in a true genetic disorder. Screening p r o g r a m s for such genetic disorders will continue to g r o w and b e c o m e m o r e comprehensive. It is i m p o r t a n t that regionalisation o f testing should begin so that large and experienced central laboratories and centres can serve as the p r i m a r y facilities. Only in this m a n n e r will neonatal screening be both effective and efficient.

REFERENCES

Berry, H. K. (1959) Procedures for testing urine specimens dried on filter paper. Clinical Chemistry, 5, 603-609. Bickel, H., Gerrard, J. & Hickmans, E. M. (1953) Influence of phenylalanine intake on phenylketonuria. Lancet, ii, 812-813. Burns, J. & Vanderlinde, R. E. (1971) False-positive test for branched-chain ketonuria. New England Journal of Medicine, 285, 753. Dent, C. E. (1946) Detection of amino acids in urine and other biological fluids. Lancet, ii, 637-639. Efron, M. L., McPherson, T. C., Shih, V. E., Welsh, C. F. & MacCready, R. A. (1969) D-Methioninuria due to DL-methionine ingestion. An artefact detected by a mass screening program for errors of amino acid metabolism. American Journal of Diseases of Children, 117, 104-107. Efron, M. L., Young, D., Moser, H. W. & MacCready, R. A. (1964) A simple chromatographic screening test for the detection of disorders of amino acid metabolism. New England Journal of Medicine, 270, 1378-1383. Ghadimi, H. (1967) Diagnosis of inborn errors of amino acid metabolism. American Journal of Diseases of Children, 114, 433-439. Guthrie, R. (1961) Blood screening for phenylketonuria. Journal of the American Medical Association, 178, 863-865. Guthrie, R. (1972) Mass screening for genetic disease. Hospital Practice, June, 93-100. Guthrie, R. & Murphey, W. H. (1971) Microbiologic screening procedures for detection of inborn errors of metabolism in the newborn infant. In Phenylketonuria and Some Other Inborn Errors of Amino Acid Metabolism. Section VII. Screening, ed. Bickel, H., Hudson, F. P. & Woolf, L. 1. Stuttgart: Georg Thieme. Guthrie, R. & Susi, A. (1963) A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics, 32, 338-343. Knox, W. E. (1960) An evaluation of the treatment of phenylketonuria with diets low in phenylalanine. Pediatrics, 26, 1-11. Levy, H. L. (1974) Genetic screening. In Advances in Human Genetics, ed. Harris, H. & Hirschhorn, K. Vol. 4, Ch. 1. New York: Plenum. Levy, H. L., Madigan, P. M. & Lure, A. (1969a) Fecal contamination in urine amino acid screening. Artifactual cause of hyperaminoaciduria. American Journal of Clinical Pathology, 51, 765-768. Levy, H. L., Shih, V. E. & Madigan, P. M. (1972) Massachusetts Metabolic Disorders Screening Program. I. Technics and results of urine screening. Pediatrics, 825-836. Levy, H. L., Shih, V. E., Madigan, P. M., Karolkewicz, V., Carr, J. R., Lum, A., Richards, A. A., Crawford, J. D. & MacCready, R. A. (1969b) Hypermethioninemia and other hyperaminoacidemias. Studies in infants in high-protein diets. American Journal of Diseases of Children, 117, 96-103. MacCready, R. A. & Hussey, M. G. (1964) Newborn phenylketonuria detection program in Massachusetts. American Journal of Public Health, 54, 2075-2081. Ntitzenadel W. & Lutz, P. (1960) Elution und diinnschichtchromatographische Auftrennung von Plasma--Aminos~.uren aus mit Blut getrfi,nkten Filterscheibschen. Clinica Chimica Aeta, 20, 151-156.

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Scriver, C. R., Davies, E. & Cullen, A. M. (1964) Application of a simple micromethod to the screening of plasma for a variety of anainoacidopathies. Lancet, ii, 230-232. Thalhammer, O. & Scheiber, V. (1972) Genetic difference between hyperphenylalaninemia and phenylketonuria. Neuropddiatrie, 3, 358-361. Turner, B. & Brown, D. A. (1972) Amino acid excretion in infancy and childhood. A survey of 200 000 infants. Medical Journal of Australia, 1, 62-65.