Newborn Screening For Phenylketonuria Research Articles

Delayed increase in blood phenylalanine concentration in phenylketonuric children initially classified as mild hyperphenylalaninemia

Introduction: Confirmatory blood phenylalanine concentrations obtained after newborn screening identification are used to establish the diagnosis of phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP). This diagnosis usually determines whether dietary treatment is necessary. When the phenylalanine level is in the range of MHP and diet is not given, there is a certain degree of complacency about followup beyond the first 6–12 months of age, since in MHP the levels usually remain in that range even with a normal diet. However, we have encountered infants whose blood phenylalanine levels rose very slowly from below those considered for dietary therapy to levels for which diet is advisable. Methods: Newborn screening for PKU was performed by Guthrie bacterial inhibition assay (GBIA). Followup blood phenylalanine levels were obtained in plasma by the amino acid analyzer, in serum by McCaman-Robins fluorometric assay, or in whole blood by GBIA. Results: The initial confirmatory phenylalanine levels in the four infants ranged from 3.0 to 8.4 mg/dl (180–509 μM). At ages ranging from 11 to 21 months and while on normal diets, the blood phenylalanine levels rose to a range of 16.0 to 18.5 mg/dl (970–1120 μM), and dietary treatment (in three infants) was given. Discussion: This experience emphasizes the need for frequent and consistent followup of the blood phenylalanine levels of infants classified as having MHP and not given dietary treatment. This also emphasizes the potential value of genotyping the phenylalanine hydroxylase (PAH) gene in infants to determine the category of hyperphenylalaninemia as early as possible.

A missense mutation in a patient with guanosine triphosphate cyclohydrolase I deficiency missed in the newborn screening program

A patient with guanosine triphosphate cyclohydrolase I deficiency passed the newborn phenylketonuria screening program. The characteristic clinical phenotype developed in a 5-month-old patient; elevated plasma phenylalanine, undetectable urinary pterins, and absence of the enzyme activity in a liver biopsy were present. A point mutation that results in an amino acid substitution from methionine to isoleucine at position 211 was proposed to be the cause for this new phenotypic expression of guanosine triphosphate cyclohydrolase I deficiency. (J P EDIATR 1995;126:401-5)

Effect of specimen collection method on newborn screening for PKU

Introduction: Although heelstick followed by direct spotting of blood on filter paper is usually the method of choice for routine newborn screening of phenylketonuria (PKU) and other disorders, current National Committee for Clinical Laboratory Standards (NCCLS) recommendations provide guidelines for blood collection by capillary tube and dorsal vein, finding that these methods are acceptable alternatives. These latter methods are used for newborn blood collection by a number of hospitals in the US. In large newborn screening programs, however, standardization and cutoffs are set by large-scale testing of specimens collected by direct spotting on filter paper. Methods: Three separate groups of 21 149, 111 035, and 120 837 newborn filter paper specimens were analyzed for phenylalanine concentration by a fluorometric method to determine the effect of collection method of phenylalanine values. The three methods examined were direct spotting of blood from heelstick on filter paper, collection of blood into capillary tube with transfer to filter paper and dorsal hand venipuncture with transfer to filter paper. Results: The mean phenylalanine levels were 2.63 mg/dl, 2.46 mg/dl, and 2.28 mg/dl for direct spotting, capillary tube, and dorsal hand vein, respectively. Actual presumptive positive rates for the three methods were 23 10 000 , 11 10 000 and 0 10 000 , respectively. These differences were consistent when controlling for laboratory and Area Genetic Center (AGC) origin. Discussion: Since guidelines generally recommend or require direct spotting, values obtained by this method should remain the standard for setting cutoffs; deviations from this method could lead to missed cases, particularly for early-tested infants with borderline elevated values.

NEWBORN SCREENING FOR PKU IN SÃO PAULO — BRASIL -.

In Brazil, newborn screening for PKU started in 1976, using filter paper impregnated with blood samples drop colected from the newborns' heels. First done in São Paulo,the program progressively extended to other Brazilian cities.Blood samples (PHE)levels were determined by spectrofluorometry. Whenever the PHE levels were found to be higher than 4mg/dl, the child was recalled for a repeat test. PKU(phenylketonuria) and hyperphenylalaninemia(PHE>10mg/dl) cases were followed-up by a multidisciplinar team. From June 1976 up to July 1992, 2, 876, 548 newborns were screened:169 (1:17,020) had PKU;10 had hyperphenylalaninemia (PHE=10-14,9mg/dl)about 0,1% had transitory hyperphenylalaninemia (PH»4-10mg/dl); there was one case of dihydrobiopterin deficiency and one case of PKU with congenital hypothyroidism(the only case in the literature so far, published in Pediatr.Res.1986, 15:176). Due to the obtained results, newborn screening for PKU and congenital hypothyroidism (CH) became obligatory by law in the State of São Paulo in 1983, expanded to all the Southern states of Brazil and finally ended up in a Federal Law in October 1990.Illiteracy of the patients' relatives keeps part of them from showing for repeat tests. Often dietary directions have to be given by means of drawings. On the whole, about 1/3 of the cases with increased Phe blood levels fail to come to APAE-Saão Paulo for final diagnosis and -if necessary- further follow-up.

Twenty-five years experience with newborn screening for phenylketonuria (PKU) in Poland

Dravet syndrome is a rare epileptic encephalopathy characterized by treatment-resistant polymorphic seizures. Seizure onset usually occurs during the first year of life, and seizures are often associated with heat-related triggering factors (e.g., fever, photosensitivity, or hot bath). It has been reported that children with Dravet syndrome often present with recurrent febrile seizures and vaccination-related seizures.We analyzed the occurrence of vaccination-related seizures (defined as the development of a seizure within 48 hours post vaccination) in 54 patients with Dravet syndrome. Patients were divided into two groups according to whether seizures occurred within 48 hours of vaccination (i.e., vaccination-proximate group) or not (vaccination-distant group).There was no significant difference in the vaccination-proximate group and vaccination-distant group for the presence of SCN1A mutation. In our Dravet syndrome cohort, the vaccination-proximate group consisted of 17 (31.5%) patients with Dravet syndrome. Thus vaccination-related seizures are a common triggering factor in Dravet syndrome, reported in up to one third of our patients.Vaccination-related seizures may act as the triggering factor for the onset of seizures in children with Dravet syndrome, especially before the definitive diagnosis of Dravet syndrome can be made within the first year of life. We suggest further study of guidelines and protocols for the prevention and management of vaccination-related seizures in children with recurrent febrile seizures pending a definitive diagnosis of Dravet syndrome in the first 12 months of life.

Microassay system for newborn screening for phenylketonuria, maple syrup urine disease, homocystinuria, histidinemia and galactosemia with use of a fluorometric microplate reader

Microfluorometry for measuring phenylalanine, branched-chain amino acids, homocysteine and histidine in dried blood spots is presented as a new mass screening method for phenylketonuria, maple syrup urine disease, homocystinuria and histidinemia, respectively. Simple and rapid determinations of the metabolites are achieved by adapting each of the optimum fluorogenic reactions to a microplate scale followed by using a highly sensitive microplate reader. Each assay for several hundreds of samples can be completed within 3 h, with fully calculated results by an on-line microcomputer. The proposed system, both with simple procedure and quantitative results, is useful for the multiple routine screening for the four aminoacidopathies, in addition to galactosemia as we reported earlier [20]. (1) The usage of a microplate system for routine microplate screening might be able to open a new style of mass-screening. (2) Measurement of homocysteine seems to be more desirable than measuring methionine in order to detect classic homocystinuria, and to enable detection of the remethylatine defects. However, the recovery procedure of homocysteine needs to be improved; furthermore, the measuring of three branched-chain amino acids will be better than determining only leucine for detecting a mild form of maple syrup urine disease or other conditions which increase the levels of these amino acids. The manner in which we measure phenylalanine in our method is more sensitive than others using the microplate system.

Maternal Phenylketonuria

Prior to newborn screening and the availability of dietary treatment for phenylketonuria, most patients with classic phenylketonuria developed profound mental retardation and rarely reproduced.1,2 Newborn screening for phenylketonuria has been routinely available throughout the United States for approximately 25 years. Many infants who were identified as having elevated blood phenylalanine concentrations received dietary treatment. In most cases they have attained normal intelligence. A significant number of women who are no longer being treated have entered or are approaching their reproductive years. When diet is discontinued, elevated blood phenylalanine concentrations and other biochemical changes of classic phenylketonuria again become evident. Children born to mothers whose blood phenylalanine concentrations exceed 0.6 mM (10 mg/dL) have an extremely high incidence of microcephaly and mental subnormality due to the teratogenic effects of phenylalanine and its metabolites.1-8 The incidence of other congenital anomalies, such as congenital heart defects, is also increased. When blood phenylalanine concentrations are less than 0.6 mM(10 mg/dL), the effect on offspring appears to be less severe and less constant. The outcome of such pregnancies is being investigated in a collaborative study sponsored by the National Institutes of Health.1,7 Additional data will become available soon. All women with persistent phenylalanine elevations should be counseled prior to pregnancy about the possible adverse effect of their condition on their offspring. These women should also be counseled about the risk that their child might have phenylketonuria (approximately 1/120). Reproductive options should be discussed. Women who decide to proceed with pregnancy should be advised that dietary treatment may improve the outcome for their child.2-4,7-9

Genetic Screening: Triumphs, Problems, and Controversies

As genetic screening becomes more widespread, it becomes increasingly important to analyze the manifold implications of genetic screening programs. This paper characterizes the various types of programs and discusses some of the scientific, ethical, social, and economic issues that arise in evaluating any genetic screening program. Two examples of successful programs, newborn screening for phenylketonuria and carrier detection for Tay-Sachs disease, are presented. We then discuss three other screening programs that have not yet been fully implemented but which have already engendered a great deal of controversy: mass screening for heterozygosity for cystic fibrosis, DNA fingerprinting in the criminal justice system, and genetic screening in the workplace.

Computerized tracking for newborn screening and follow-up: A review

In the third decade of newborn screening for phenylketonuria (PKU) and other disorders computers are being used increasingly for both the laboratory and the follow-up aspects of screening programs. In 1984 slightly less than 40% of the state programs had automated follow-up. Lack of funding is probably the major inhibitor of more widespread use of computers in tracking newborns through the newborn screening process. It is suggested that federal funds be made available to ensure wider distribution of currently used tracking systems and development of methods for tracking newborns from birth through follow-up.

1234 RELIABILITY OF THE GUTHRIE BACTERIAL INHIBITION ASSAY (BIA) FOR PHENYLALANINE

The BIA is the primary method used in newborn screening for phenylketonuria (PKU). To test the reliability of the BIA to differentiate known phenylalanine (phe) standards, seven subjects experienced with BIA for newborn screening measured growth diameters of 18 phe standards distributed randomly on 6 plates. These standards included 2, 4, 6, 8, 10, 12, 20 mg/dl. Each concentration was represented in duplicate or triplicate and no concentration was replicated on the same plate. Six subjects showed overlap between standards of different concentrations, and serial pairs of standards all showed some degree of overlap. Comparisons within individual subjects indicated 3 subjects with ≥ 1/3 and 4 subjects with > 1/3 to 2/3 overlap. Overlapping measurements were noted between each serial pair of standards and the frequency of overlap ranged from 5-51%. There were 8 serial phe pairs with both members of the pair appearing on the same plate; there was 43% overlap on one pair, 14% overlap on 3 pairs and no overlap on the remaining 4 pairs. We conclude that there is considerable overlap in measurements of diameters of bacterial growth using the BIA throughout clinically relevant concentrations. This may account for a portion of the false negative and false positive PKU screens. Our limited subject population showed wide variability in individual performances. A test of performance might be appropriate in selection of personnel involved in newborn screening by BIA.

Neonatal Screening for Phenylketonuria

In Reply.— The comments of Koch and Twelmeyer and of Berlow are appreciated as they allow us to reemphasize points made in our recent article1 on newborn screening for phenylketonuria (PKU) and permit us to address additional relevant issues. Koch and Twelmeyer have stated that "only two of the 26 countries reported false-negative results involving the Guthrie test" and therefore these false-negative results "could be treated as outliers" affecting "the conclusion reached as a result of this survey."

Neonatal Screening for Phenylketonuria

To the Editor.— Publication of the article by McCabe et al1 in September 1983 marks the twentieth anniversary of Guthrie and Susi's description of newborn screening for phenylketonuria (PKU), also published in these pages.2 The bacterial inhibition assay (BIA), which was reported in the latter article became the basis of a system of preventive pediatric care which has expanded during the past two decades. In 1974, Holtzman et al3 were the first to examine extensively the issue of false-negative tests or missed PKU, and this problem has become a major concern.

Newborn Screening for Phenylketonuria: Predictive Validity as a Function of Age

Data from questionnaires were assembled for 109 infants with phenylketonuria (PKU) and 114 control infants to assess the predictive validity of newborn screening for PKU as a function of age. Patients with PKU had values of less than 4 mg/dL in cord blood and in samples from days 1, 2, and 4 through 7. The proportion of patients with PKU expected to fall below screening cutoffs of 2, 4, and 6 mg/dL was predicted for each age range. Using a cutoff of 4 mg/dL, approximately one third of patients with PKU would be missed by a sample taken from the neonate in the first 12 hours of life, and nearly 10% would be missed with a sample from the second 12 hours of life. This study shows that not all patients with PKU will be detected by newborn screening, and that the phenomenon of early nursery discharges must be considered in developing appropriate screening strategies.

Newborn Phenylalanine/Tyrosine Metabolism

Controversy over the sensitivity of newborn screening for phenylketonuria (PKU) has recently led to the specific recommendation that infants discharged within the first 24 hours of life be routinely retested at 1 to 2 weeks of age. To address this controversy, data from cord blood samples, phenylalanine (Phe) tolerance tests at 3 days of age, and sequential plasma Phe determinations in six siblings of known cases of PKU or hyperphenylalaninemia were analyzed, along with data from the literature. These analyses showed that normal and probably heterozygous infants decrease their Phe concentrations with age, while infants with PKU or PKU variants increase their Phe concentrations with age but at varying rates. Predictive equations showed that the sensitivity of the screening test rapidly increases with age, so that the usual critical value of 4 mg/dL is satisfactory for suspecting PKU after 18 hours of age. A critical value of 3 mg/dL would be equally satisfactory at any age, if the Guthrie test were appropriately modified to include a 3-mg/dL standard.

New Issues in Newborn Screening for Phenylketonuria and Congenital Hypothyroidism

Screening for phenylketonuria (PKU) and congenital hypothyroidism (CH) is of concern to parents, physicians, and public health professionals. Parents have an abiding interest in a predictive activity that can prevent disease in their offspring. Physicians and their consultants must counsel parents and interpret a positive screening test. Public health personnel are concerned with the specificity and sensitivity, efficiency, and effectiveness of newborn screening. The Committee on Genetics has previously published recommendations on newborn screening for PKU and CH.1 Further recommendations are required because PKU and CH screening are widely practiced joint procedures in the newborn and because most full-term newborn infants are now being discharged from North American nurseries within the first three days after birth. This new practice is likely to have an effect on the validity of newborn screening and on screening for PKU, in particular. A recent statement from the Committee on Fetus and Newborn (Pediatrics 65:651, 1980) addressed the problem of so-called "in and out deliveries." However, the statement was ambiguous on the issue of whether such infants should be screened routinely on the initial discharge from the nursery. The Committee on Genetics believes that all infants, regardless of age, should be screened for PKU and CH at discharge from the nursery. The Committee believes that screening is not the equivalent of diagnosis; some cases of PKU and CH will inevitably be missed by screening. Whereas an important reason for missed cases may be the biology of the target disorders, none should slip through the screening mesh because of flaws in the program and its components.

An evaluation of routine follow-up blood screening of infants for phenylketonuria.

ROUTINE follow-up blood testing has been advocated for newborn phenylketonuria screening programs.1 2 3 This advocacy derives from the fact that an occasional neonate with the disorder is undetected in routine screening programs that generally use a single blood specimen obtained during the first days of life4 and from the assumption that these infants would be detected if a second or follow-up blood specimen were obtained several weeks later. It is assumed that such affected neonates are not identified from the initial blood specimen because this specimen may be obtained before enough protein has been ingested to result in demonstrable hyperphenylalaninemia.1 These . . .

Regional Newborn Screening for Hypothyroidism

Newborn screening for phenylketonuria (PKU) is now so firmly established in North America and in many other areas of the world that it is a bit difficult for most pediatricians to recall when PKU was diagnosed on the basis of mental retardation. The key to PKU screening has been the Guthrie test, the genius of which is not the bacterial assay that is employed (though this assay was certainly a momentous development) but the simple filter paper blood specimen. This specimen, easily obtained by blotting a few drops of blood from the heel of a neonate, is even more easily mailed to a central laboratory for testing.

Experience with Testing for Phenylketonuria in Massachusetts

Because it seems unfortunate that misunderstandings in some places have delayed the inauguration of screening programs for phenylketonuria (PKU) in newborns, I am sending this or a similar letter to the editors of five or six medical journals. It occurred to me that a preliminary published note regarding the 10-month experience of Massachusetts in the nation-wide PKU newborn screening project might be of value to physicians and health officers of other states, especially in states not presently participating or participating only on a limited basis.