Newborn Screening For Phenylketonuria Research Articles

Historical Perspectives

It began with Bob Guthrie’s second child. John was mentally retarded. Because of this, Guthrie was stimulated toward research that could prevent mental retardation and other developmental disabilities. In 1957, Guthrie was conducting cancer research at the Roswell Park Cancer Institute in Buffalo, NY. Because of John, Bob and his wife Margaret were very active in the Buffalo chapter of the New York State Association for Retarded Children. As Vice-President of the chapter, he was responsible for the program at the monthly meeting. He had heard that Dr Robert Warner, Director of the newly established Children’s Rehabilitation Center at Buffalo Children’s Hospital, was doing work in developmental disabilities, so he invited Dr Warner to speak about his work at one of the meetings. At the meeting, Dr Warner discussed his work in treating children with mental retardation, some of whom had metabolic disorders. Over coffee after the meeting, Guthrie mentioned his interest in the prevention of mental retardation. As a result of the meeting and especially the discussion after the meeting, the two kept in touch. About a year later, Dr Warner introduced Guthrie to phenylketonuria (PKU). Dr Warner explained that PKU was an inborn error of phenylalanine metabolism that caused mental retardation, but that when these retarded children received a special phenylalanine-restricted diet, not only did their blood phenylalanine levels decrease, but their behavior improved substantially. One problem in administering the diet, Dr Warner explained, was the difficulty with tests required to measure the blood phenylalanine levels for close monitoring of the patients. This persuaded Guthrie to try to produce a simpler method for measuring blood phenylalanine. Guthrie thought such a test might be possible by modifying the Bacillus subtilis or Escherichia coli bacteria assays he was using to screen for different antimetabolites in blood from patients who were …

High protein diet mimics hypertyrosinemia in newborn infants

Tyrosinemia resulting from administration of protein-dense infant diets was detected by newborn screening in two infants. Change of formula resulted in rapid resolution of the hypertyrosinemia. These cases identify nonstandard infant diets as a benign and reversible cause of tyrosinemia and a potential cause of positive newborn phenylketonuria screening.

Behavioural factors related to metabolic control in patients with phenylketonuria

The objective of this study was to determine the importance of parental factors possibly related to dietary control in early and continuously treated patients with phenylketonuria (PKU). A questionnaire was disseminated among parents of 238 patients with PKU born after the nationwide introduction of newborn screening for PKU (1 September 1974) until 31 December 1995. The questionnaire was based on a behavioural model measuring people's attitudes, subjective norms, and self-efficacy. Dietary control was defined on the basis of mean phenylalanine (Phe) concentration of the PKU patients measured between 1 January 1994 and 31 December 1996. Response rate was 71%. Attitudes: children of parents who believed that their child adheres well to the diet, even if his or her Phe concentrations are sometimes too high, had lower Phe concentrations than children of parents who disagree with this statement (adjusted difference -103 micromol/L, p < 0.001). Subjective norm: Phe concentrations were higher when parents answered that their relatives did not approve when their child deviates from the diet (p = 0.004). Self-efficacy: children of parents who reported difficulties in having their child eat the synthetic protein substitute three times a day had higher Phe concentrations than those of parents who did not have such difficulties (adjusted difference 156 micromol/L, p = 0.007). More attention should be given to parents having their child eat the synthetic protein substitute at least three times a day and to teaching parents to keep strictly to the diet without being too rigid. These factors were strongly associated to dietary control and may be amenable to change.

Recent advances in newborn screening for neurometabolic disorders.

Newborn screening for neurometabolic disorders offers a unique and promising opportunity to practice preventive medicine in children with diverse inborn errors of metabolism. The benefits of the early identification and presymptomatic treatment of these disorders are now recognized. The rapid pace of developments in the field of expanded newborn screening has been made possible by technological advances in the ability to detect multiple compounds diagnostic of diverse inborn errors of metabolism in neonatal dried blood specimens. Launched by the success of newborn screening for phenylketonuria, experts in this area are optimistic that further public health benefits will follow the same pathway. This review highlights the population model and public health issues inherent in advances in newborn screening for selected neurometabolic disorders. Current and future diagnostic and therapeutic, as well as social and ethical, dilemmas are discussed. Newborn screening represents one of the major child health advances of this past century. As developments in the field of expanded newborn screening for neurometabolic disease progress forward, the long-term success of this important project will pose future challenges and opportunities.

Community-Based Program for the Diagnosis and Prevention of Genetic Disorders in Cuba

The author’s experience of 20 years as director of the medical genetic services program in Cuba is presented. The setting of the infrastructure for equipment and the training of personnel for the medical genetic program began in 1981 in the city of Havana, and was progressively extended to cover the whole country in 1988. Between 1982 and 2002, 2.8 million pregnant women were tested for sickle cell carrier status, 96,000 carriers and 4,786 couples at risk were detected and offered genetic counseling and prenatal diagnosis. In the same period, the combination of maternal serum α-fetoprotein screening and fetal ultrasound led to the prenatal diagnosis of anomalies in several thousand fetuses. The accessibility to legal abortion, and the autonomous decisions by the majority of couples to terminate abnormal pregnancies reduced the prevalence of neural tube defects and sickle cell disease at birth by 90 and 65%, respectively, by 2002. In the span of approximately 20 years, 22,690 pregnant women at risk received prenatal chromosomal diagnosis. Newborn screening for phenylketonuria and congenital hypothyroidism was established. Genetic counseling was offered to every detected person or family at risk for genetic conditions. The network of medical genetic services established in the country received a very positive acceptance by the population. A very successful connection with the primary health care level was established.

Long-term follow-up of a patient with mild tetrahydrobiopterin-responsive phenylketonuria

We report on the long-term follow-up of the first Italian patient with the tetrahydrobiopterin (BH 4)-responsive type of phenylalanine hydroxylase deficiency (R243X/Y414C genotype). The patient was diagnosed by the newborn screening for phenylketonuria (PKU) and with a positive BH 4 loading test. Introduction of BH 4 (initially 10 and later 20 mg/kg/day) in addition to reduced low-phenylalanine diet resulted in therapeutic plasma phenylalanine concentrations (<340 μmol/L). Very good compliance and no side effects in this patient demonstrate the great potential of BH 4 in the treatment of some patients with mild PKU.

Categorizing genetic tests to identify their ethical, legal, and social implications.

Practice standards in medical genetics provide an implicit guide to the ethical, legal, and social implications (ELSI) of genetic tests. The common use of nondirective counseling reflects the principle that many testing decisions should be determined by personal values. Yet geneticists make test recommendations in some circumstances, e.g., RET mutation testing for MEN2 and newborn screening for phenylketonuria (PKU). Conversely, many geneticists recommend against testing for Apolipoprotein E (ApoE) alleles to predict Alzheimer disease (AD) risk. Taken together, these examples suggest that genetic tests can be categorized by a joint consideration of clinical validity and availability of effective treatment for persons who test positive. For genetic tests with high clinical validity/no treatment (e.g., presymptomatic testing for Huntington disease), the predominant concern is adequate nondirective counseling to ensure an informed, autonomous decision. By contrast, the predominant concern for tests with high clinical validity/effective treatment (e.g., PKU) is assuring access to care for eligible persons. For tests with limited clinical validity/no treatment (e.g., ApoE), recommending against test use can be justified on the principle of avoiding harm. For a fourth category, tests with limited clinical validity/effective treatment (e.g., HFE mutation testing for hereditary hemochromatosis), net benefit is the issue: the balance between potential benefits of treatment and potential harms of genetic labeling must be weighed. Where uncertainty exists concerning both clinical validity and effectiveness of treatment, as in the case of BRCA 1/2 mutation testing, the value of testing may vary according to different testing contexts. This approach to test categorization allows a rapid determination of the predominant ELSI concerns for different kinds of genetic tests and identifies the data most urgently needed for test evaluation.

Newborn Screening by Tandem Mass Spectrometry

After completing this article, readers should be able to: 1. List examples of disorders for which tandem mass spectrometry (MS/MS) can screen. 2. Delineate potential difficulties with MS/MS newborn screening. Tandem mass spectrometry (MS/MS) technology has shown tremendous promise in newborn screening pilot programs worldwide with its capacity to measure numerous metabolites from a dried blood spot virtually simultaneously with an extremely rapid throughput per sample (approximately 2 min). The expansion of newborn screening programs by inclusion of an additional 15 to 30 metabolic disorders has the potential to decrease significantly the morbidity and mortality associated with inborn errors of metabolism and could offer the clinician critical diagnostic information when caring for an acutely ill neonate. Newborn screening for metabolic disorders began in 1962 in the United States, with the introduction of the Guthrie bacterial inhibition assay for phenylketonuria (PKU) in a Massachusetts voluntary program. Child health advocates, including the National Association for Retarded Citizens and the March of Dimes Birth Defects Foundation, developed model legislation and lobbied for passage of newborn screening laws at the state level. As a result of these efforts, newborn screening for PKU was legally mandated in most states in the early 1960s. The success of newborn screening for PKU led to development of tests for other conditions, including metabolic disorders (eg, galactosemia, maple syrup urine disease [MSUD], homocystinuria, and biotinidase deficiency), endocrinopathies (eg, congenital hypothyroidism and congenital adrenal hyperplasia), hemoglobinopathies (eg, sickle cell disease and thalassemias), and cystic fibrosis. Different states screen for a variable number of these disorders, with most screening for three to six conditions (Figure⇓ ). Such differences reflect state political and economic environments, technologic capabilities of public health departments, regional ethnic composition, and community expectations. All states screen for PKU and congenital hypothyroidism, and 48 states screen for galactosemia. Other inborn …

Screening for the IDDM high-risk genotype. A rapid microtitre plate method using serum as source of DNA.

Norwegian babies born with the HLA-DRB1*0401-DQA1*03-DQB1*0302/DRB1*03-DQA1+ ++*05-DQB1*0201 genotype have an estimated 17% lifetime risk of developing insulin-dependent diabetes mellitus (IDDM). Identifying these children is important for future prevention, and for studies of the non-genetic factors involved in IDDM. The aim of the study was to develop a rapid screening method for this high-risk genotype. DNA was extracted from serum collected during routine newborn screening for phenylketonuria and hypothyreosis. The second exons of HLA-DQA1 and DQB1 were co-amplified using biotinylated primers, amplicons were hybridized to a set of seven probes immobilized on a microtitre plate using a single hybridisation temperature, and detected colorimetrically by streptavidin-HRP conjugate and tetramethylbenzidine substrate. The DRB1*04 subtyping was performed using six different probes at identical conditions. The prevalence of the DRB1*0401-DQA1*03-DQB1*0302/DRB1*03-DQA1*0 5-DQB1*0201 genotype among 1,026 Norwegian babies was 2.7% (CI 95%: 1.7-3.7%). The new high-throughput genetic screening method for IDDM risk can easily be automated and included in newborn screening programs.

Serving the Family From Birth to the Medical Home

Newborn screening in the United States is a public health program aimed at the early identification of conditions for which early and timely interventions can lead to the elimination or reduction of associated mortality, morbidity, and disabilities. This screening takes place within the context of a newborn screening system, and involves the following components: screening, short-term follow-up, diagnosis, treatment/management, and evaluation. Inherent to each of these components is an education process. The screening programs like these for the 4 million infants born each year in the United States have been heralded as successful and cost-effective.1–5 The newborn screening program's efficiency and effectiveness depends on the smooth integration of sample collection, laboratory testing, follow-up, diagnosis, timely treatment, and tracking of outcomes.6–11 The foundation and justification of newborn screening systems rest on the principles that testing procedures are readily available; technically feasible; economically sound; and clearly beneficial to affected newborns, their families, and to society.10,12–14 The universal acceptance of newborn screening for specified conditions over the past 3 decades attests to the undeniable benefits that flow from early testing and prompt, appropriate therapy. However, although newborn screening systems have succeeded in preventing morbidity and mortality, controversies, challenges, and opportunities continue. ### The History of Newborn Screening Newborn screening programs began in the early 1960s with the original work of Dr Robert Guthrie, who developed a screening test for phenylketonuria (PKU) and a system for collection and transportation of blood samples on filter paper.15,16 By 1962, Massachusetts launched a voluntary newborn PKU screening program that demonstrated the feasibility of mass genetic screening.17 Initially, newborn screening for PKU was not a health department role or a legislated activity. Health professionals were slow to adopt the practice of screening for PKU, and the responsibility for screening was not defined (eg, should it be the responsibility of …

Maternal Phenylketonuria: A New Cause for Concern

The start of newborn screening for phenylketonuria (PKU) during the early 1970s has given rise to an increasing number of women who have been identified and successfully treated for the disease in childhood and are now preparing to have children of their own. Early detection and initiation of nutritional therapy before conception is key to a successful pregnancy outcome. Nurses who understand the pathophysiology, dietary limitations, and management of PKU in pregnancy can provide the care necessary for optimal maternal and neonatal health.

Phenylalanine and Tyrosine Quantification by Stable Isotope Dilution Liquid Chromatography–Mass Spectrometry from Filter Paper Blood Spots

Phenylketonuria (PKU) is one of the more common inborn errors of metabolism, affecting 1 in 10 000–15 000 individuals (1). Newborn screening for PKU is performed in all 50 states and started with the development of the bacterial inhibition screening assay by Guthrie and Susi (2). Frequent monitoring of blood Phe concentrations is required to maintain the concentration as close as possible to the reference interval [60–120 μmol/L (1–2 mg/dL)] to prevent damage to the brain from increased Phe concentrations and to prevent a negative nitrogen balance from an overly Phe-restricted diet. Moreover, the high risk of birth defects for the fetus of a PKU mother requires extremely frequent monitoring of blood Phe concentrations and would greatly benefit from a rapid and accurate blood spot method. Phe determination from dried blood spots is feasible by various methods: bacterial (2), fluorometric (3), liquid chromatography (LC) (4), and tandem mass spectrometry (MS) (5). Of these methods, only tandem MS allows use of a stable isotope as internal standard. Although tandem MS is the most rapid method available, the extremely high equipment cost and the need for sample derivatization are disadvantages. We developed a rapid and precise blood-spot method for Phe and Tyr determination using stable isotope dilution reversed-phase LC-MS. The advantages of this method are the very short sample preparation without derivatization, a short chromatographic run, the use of selective detection to eliminate interference, and the use of ideal internal standardization (stable isotope). The method described herein is now used routinely in our PKU clinic. l-Phe ( M r 165.19) and l-Tyr ( M r 181.19) were purchased from Sigma Chemical Co. Stable isotopes [ring-d5]-l-phenylalanine ( M r 170.23) …

Present newborn screening for phenylketonuria

Newborn screening for phenylketonuria (PKU) began in the United States in the early 1960s following development of the Guthrie bacterial inhibition assay that allowed for the easy, rapid screening of elevated blood phenylalanine levels collected on newborn filter paper samples. Since that time, a number of other techniques that screen for PKU, other inborn errors of metabolism, and a variety of nonmetabolic disorders have been developed using newborn blood samples. The most advanced, comprehensive technique available today is tandem mass spectrometry (MS-MS), which simultaneously identifies and measures many compounds of varying structural classes allowing for concurrent screening of many disorders, including aminoacidemias, organic acidurias, and fatty acid disorders. Despite this progress in presymptomatic neonatal detection of PKU, there are some difficulties with newborn screening. These include false-positive results, occasional missed diagnoses, and problems surrounding early discharge. In addition, not all elevated phenylalanine levels are a result of a deficiency of the liver enzyme phenylalanine hydroxylase (PAH). Some infants have transiently elevated levels. Others have defects in the synthesis or recycling of tetrahydrobiopterin (BH4), the cofactor of PAH, and some have secondary phenylalanine elevations due to disorders that affect the liver, such as tyrosinemia or galactosemia. Several of these problems, including the number of false positives, may be eliminated by pattern identification via MS-MS. Once identified with a significant or persistent elevation in phenylalanine, infants and their families are referred to a metabolic center for further evaluation, including repeat quantitative testing and, if necessary, dietary or cofactor therapy. MRDD Research Reviews 1999;5:144–149. © 1999 Wiley-Liss, Inc.

Use of phenylalanine-to-tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours

We compared the screening interpretation of fluorometric analytical results for phenylketonuria (PKU) with tandem mass spectrometry (MS/MS) in filter paper blood spots collected from newborns <24 h of age. In MS/MS, both Phe and Tyr are quantified. Two hundred and eight blood spots collected from infants <24 h of age were retrieved from storage from the California newborn screening program. These samples had been categorized on the basis of fluorometric analysis as initial negative, initial positive for hyperphenylalaninemia with negative determination on recall, or initial positive for hyperphenylalaninemia and confirmed on follow up as PKU or variant hyperphenylalaninemia. The retrieved samples were analyzed in a blinded fashion using MS/MS. Correlation analysis of fluorometry vs MS/MS for Phe concentration was high, with a Pearson correlation coefficient of 0.817. When 180 micromol/L was used as the cutoff Phe concentration for MS/MS and 258 micromol/L was used as the cutoff for fluorometry, all infants with confirmed classical PKU and variant hyperphenylalaninemia were detected. MS/MS analysis reduced the number of false-positive results from 91 to 3. Simultaneous quantification of Phe and Tyr by MS/MS with the use of a cutoff Phe/Tyr molar ratio of 2.5 further reduced the number of false positives to 1. Samples from affected infants showed a discernible trend of increasing Phe concentration and Phe/Tyr molar ratio with age of collection. These results demonstrate the utility of MS/MS in the routine PKU screening of early-discharge newborns. MS/MS reduces the false-positive rate of fluorometric screening almost 100-fold because of the improved accuracy and precision of Phe measurement and simultaneous confirmation with the Phe/Tyr molar ratio. In addition to the detection of PKU, MS/MS can also detect other aminoacidopathies and disorders of fatty acid and organic acid metabolism with lower false-positive rates than other methods currently used in newborn screening programs.

A systematic review of evidence for the appropriateness of neonatal screening programmes for inborn errors of metabolism.

Developments in screening technology and increased understanding of the natural history and treatment of inborn errors of metabolism (IEMs) have produced pressure to extend neonatal screening programmes. This review aims to assess the evidence for the appropriateness of such programmes. A formal systematic literature review was conducted. Exclusion and inclusion criteria were used to select papers for critical appraisal by pairs of reviewers. Standard criteria were used to assess the appropriateness of neonatal screening for various IEMs. Site visits were conducted to assess new technologies for newborn screening. A total of 1866 papers were identified and 407 systematically selected for full critical appraisal. Published evidence confirmed that universal newborn screening for phenylketonuria (PKU) meets all of the screening criteria and justifies the expense and infrastructure necessary for the collection and testing of neonatal blood spots. There was insufficient evidence in the literature to assess the cost-effectiveness of screening for any other IEMs. There was reasonable evidence to support inclusion in extended neonatal screening of four other IEMs: biotinidase deficiency, congenital adrenal hyperplasia (CAH), medium-chain acyl CoA dehydrogenase (MCAD) deficiency and glutaric aciduria type 1 (GA1). Large-scale trials of screening for biotinidase, CAH, MCAD and GA1 should be conducted, with careful evaluation to establish their clinical effectiveness and cost-effectiveness in practice. Screening for the latter two disorders would be dependent upon the use of tandem mass spectrometry (tandem MS). The application of tandem MS to newborn screening requires further evaluation. The extension of neonatal screening programmes to other IEMs is not currently justified.

Mild Hyperphenylalaninemia and Heterozygosity of the Phenylalanine Hydroxylase Gene

Newborn screening for phenylketonuria (PKU) is now the standard of practice. Initial phenylalanine blood levels of 240 μmol/L result in referral of affected newborns to medical facilities experienced in caring for patients with metabolic disorders. This case report concerns a female infant born in 1976 with a presumptive positive PKU screening test on the third day of life of 240 μmol/L phenylalanine. Follow-up levels while the mother was breast feeding on the sixth day of life were 324 and, on the 27th day, 312 μmol/L. She was subsequently lost to follow-up at age 11 years, but returned at 19 years of age due to pregnancy, with a blood phenylalanine level of 132 μmol/L. Mutation studies then were performed documenting that she was a carrier for the phenylalanine hydroxylase gene and did not have hyperphenylalaninemia. The mother's parents and the infant were also genotyped confirming heterozygosity. The infant on follow-up is completely normal, following a normal pregnancy.

Receiver operating characteristic plots to evaluate Guthrie, Wallac, and Isolab phenylalanine kit performance for newborn phenylketonuria screening

We used ROC plots to evaluate the clinical performance of the Guthrie, Wallac, and Isolab assays for newborn phenylketonuria (PKU) screening and assessed the screening discriminatory power of these three assays by the area under the ROC plot, Youden's J index, and the likelihood ratio. The use of these plots not only allows us to pinpoint the exact cutoff value in screening, but also provides a direct comparison of these three different assays in clinical outcome performance. The optimum cutoff for the newborn PKU screening is a blood phenylalanine concentration of 0.30, 0.27, and 0.18 mmol/L for the Guthrie, Wallac, and Isolab assays, respectively. We conclude that the Wallac and Isolab kits, like the Guthrie assay, are suitable for newborn PKU screening.

Neonatal screening for phenylketonuria and congenital hypothyroidism in Principado de Asturias (Spain) using two types of blood samples

Introduction: Newborn screening for phenylketonuria (PKU) and congenital hypothyroidism (CH) is usually conducted with a single specimen collected from the infant prior to hospital discharge. To meet the specific medical infrastructural requirements in the Principado de Asturias in northern Spain, we modified specimen collection by obtaining an umbilical cord blood specimen for CH screening and a newborn blood specimen for PKU screening from each infant. Methods: The umbilical cord blood specimen is collected on filter paper and at hospital discharge is given to the parent together with a kit for collection of the newborn blood specimen. The latter specimen is collected at age 5–8 days at a Public Health Center and mailed with the umbilical cord specimen to the screening laboratory. CH screening is performed by TSH assay and PKU screening by phenylalanine assay, which was initially performed by thin-layer chromatography (TLC) and now by fluorometric technique. Confirmatory CH testing was conducted in the newborn blood specimen by radioimmunoassay for thyroxine (T4). Results: Among 91 529 neonates tested for CH, 27 cases were detected, a frequency of 1:3,400. Females accounted for 82% of the cases. Among 75 488 tested for PKU, five cases (1:15 000) were detected. Discussion: Utilization of both umbilical cord blood and neonatal blood specimens has accommodated the health care organization of our region and allowed for comprehensive screening for CH and PKU. This strategy could be a model for other areas of the world with infrastructural conditions similar to those we have had to overcome.

Postnatal clearance of maternally derived phenylalanine in offspring of maternal phenylketonuria: implications for newborn screening

Introduction: In maternal phenylketonuria (PKU), phenylalanine transverses the placenta, producing hyperphenylalaninemia in the fetus. This hyperphenylalaninemia is present in the offspring at birth and could produce false positive newborn PKU screening results unless rapidly cleared. Consequently, we determined the duration of postnatal hyperphenylalaninemia among offspring of maternal PKU/mild hyperphenylalaninemia. Methods: Capillary blood specimens were collected from offspring of maternal PKU/hyperphenylalaninemia pregnancies within the first 24 h of life. These specimens were analyzed by Guthrie bacterial assay for phenylalanine. Umbilical cord blood specimens were collected and analyzed for amino acids by the amino acid analyzer. Results: The mean blood phenylalanine levels in 51 unaffected offspring decreased from 8.2 ± 6.1 mg/dl (497 ± 370 μmol/1) at age 4 h to 1.8 ± 0.5 mg/dl (110 ± 30 μmol/1) at age 24 h. At 12 h of age, however, the phenylalanine level was still > 2 mg/dl (120 μmol/1) in 59% and > 4 mg/dl (240 μmol/1) in 37% of these infants. Based on umbilical cord blood valves, 74 ± 22% of transferred phenylalanine remained in the offspring at 4 h of age, but the range was 32–100%, indicating wide variation in the initial clearance abilities of these infants. Two infants with mild hyperphenylalaninemia had persisting high blood phenylalanine at 24 h of age. Discussion: Most infants from maternal PKU pregnancies clear the maternally derived phenylalanine by 24 h of age. Thus, there is only a very short exposure to hyperphenylalaninemia postnatally in the infants from maternal PKU/mild hyperphenylalaninemia pregnancies. In addition, they are not likely to be designated as false positive in newborn screening, although they may be so designated in specimens collected before 24 h of age.

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.