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

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Delayed increase in blood phenylalanine concentration in phenylketonuric children initially classified as mild hyperphenylalaninemia Cheston M. Berlin Jr.*a, Harvey L. Levyb, William B. Hanley” “Department

o$ Pediatrics, The University College of bChildrenS Hospital and ‘The Hospital

Milton S. Hershey Medical Center of The Pennsylvania Stare Medicine, P. 0. Box 850. Hershey, PA 17033, USA The Harvard Medical School, Boston, MA 02115, USA for Sick Children, Toronto, Ontario, Canada

Received 9 December 1994;revisionreceived23March 1995;accepted 27March 1995

Abstract Introduction: Confirmatory blood phenylalanineconcentrationsobtained after newborn screeningidentification areusedto establishthe diagnosisof phenylketonuria(PKU) or mild hyperphenylalaninemia (MHP). This diagnosisusually determineswhetherdietary treatment is necessary. Whenthe phenylalaninelevel is in the rangeof MHP anddiet isnot given, there is a certaindegreeof complacencyabout followup beyondthe first 6- 12monthsof age,since in MHP the levelsusuallyremainin that rangeeven with a normaldiet. However, we have encounteredinfantswhosebloodphenylalaninelevelsrosevery slowlyfrom belowthoseconsideredfor dietary therapy to levelsfor which diet is advisable.Methods:Newbornscreening for PKU was performed by Guthrie bacterial inhibition assay(GBIA). Followup blood phenylalaninelevels were obtained in plasmaby the amino acid analyzer, in serumby McCaman-Robinsfluorometricassay,or in wholeblood by GBIA. Results: The initial confirmatory phenylalaninelevelsin the four infants rangedfrom 3.0 to 8.4 mg/dl (180-509PM). At agesrangingfrom 11to 21 monthsand while on normal diets, the blood phenylalanine levelsroseto a rangeof 16.0to 18.5mg/dl (970-l 120PM), and dietary treatment(in three infants)wasgiven. Discussion: This experienceemphasizes the needfor frequentand consistent followup of the blood phenylalaninelevelsof infants classifiedashaving MHP and not givendietary treatment.Thisalsoemphasizes the potentialvalueof genotypingthe phenylalanine hydroxylase(PAH) genein infants to determinethe categoryof hyperphenylalaninemia as early aspossible. Keywords: Newborn screening;Phenylketonuria;PKU; Treatment

*Corresponding author,Tel.: +I 7175318006; Fax: +I 7175318985. 0 1995ElsevierScience IrelandLtd. All rightsreserved 0925-6164/95/$09.50 SSDI 0925-6164(95)00105-U

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1. Introduction Phenylketonuria (PKU) is an inborn error of metabolism caused by the absence or an inactive form of phenylalanine hydroxylase, the enzyme that catalyzes the biotransformation of phenylalanine to tyrosine. The major biochemical effect of this enzyme deficiency is an increase in the level of phenylalanine. All 50 states in the US and all 10 provinces in Canada have newborn screening programs for the detection of increased phenylalanine to identify affected infants and provide the earliest possible dietary intervention [1,2]. As a result of these programs, most infants with PKU are diagnosed and begun on treatment before 3 weeks of age. However, these programs also identify infants with less severe degrees of hyperphenylalaninemia than the levels of 15-20 m&d1 (907-1210 PM) usually considered to represent PKU. The lower degree of hyperphenylalaninemia, usually termed mild hyperphenylalaninemia (MHP), is often considered benign and not treated [3-51. Nevertheless, the precise degree of hyperphenylalaninemia at which dietary restriction of phenylalanine should be instituted is still unclear. At the Hershey Medical Center a blood phenylalanine level of 14 mg/dl (847 FM) was previously considered necessary for dietary therapy but this level has recently been lowered to 10 mg/dl (600 PM). At Children’s Hospital in Boston the level for treatment has been 12 mg/dl (726 FM), but has now been lowered to 8 mg/dl (480 PM). At the Hospital for Sick Children in Toronto the maximum level allowed before diet initiation is 10 mg/dl (600 FM). Recently, Costello et al. [6] have recommended maintaining blood phenylalanine levels below 6.5 mgldl (400 PM). In essentially all centers the confirmatory blood phenylalanine level, obtained when the infant detected by newborn screening receives metabolic evaluation, establishes the diagnosis of PKU or MHP and determines whether dietary treatment will be given, This level is generally considered definitive. Blood phenylalanine levels in the MHP range, for instance, usually remain in that range on a normal diet through infancy, childhood and later. In fact, this experience has led to a certain degree of complacency about MHP, often with little or no followup beyond the first 6-12 months of life. This complacency may be misguided, as shown by at least one report of an infant whose blood phenylalanine level rose from the range of MHP to that of PKU [7]. We report here four additional infants whose blood phenylalanine concentrations rose slowly from those in early infancy that were below the requirement for dietary therapy to levels for which diet was advisable. Fig. 1 illustrates their blood phenylalanine levels. 2. Case reports Patient 1 was a term infant born to a gr 10 para 10 Amish woman. The newborn screening result by Guthrie bacterial inhibition assay at age 1 day was 6 mg/dl (360 PM). Subsequent bacterial inhibition assay results were 3 mg/dl (180 PM) (age 3 months), 2 mg/dl (120 PM) and 15 mg/dl (910 PM) (age 7 months), 12 mg/dl (720 PM) (age 8 months), and 9 mg/dl (540 PM) (age 10 months). At 11 months, after a

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level of 18 mg/dl(lO90 PM) was obtained, the patient, who was exclusively breastfed, was placed on a phenylalanine restricted diet. One result at 12 months of age was ~20 mg/dl (1200 PM) but since then the phenylalanine levels have been within acceptable levels on diet (2-8 mg/dl (120-480 PM)). Psychological testing (StanfordBinet) at age 3 years showed him to be in the average range although, for cultural and linguistic reasons, psychological testing of Amish children is difficult, especially at younger ages. For instance, they do not learn English until they begin school at age 5 or 6 years, they have minimal or no exposure to ordinary objects in the usual American home, and they do not associate with American children. Patient 2, a second cousin to Patient 1, was a term infant born to a gr 2 para 2 Amish woman. The newborn screening result by Guthrie bacterial inhibition assay was 6 mg/dl (360 PM). Followup serum phenylalanine levels obtained by quantitative fluorometric analysis during the first month of life were 8.3 mg/dl(502 PM), 12.5 mgfdl(760 PM), and 7.9 mg/dl (475 FM). Multiple bacterial inhibition assay results (n = 12) during the first 19 months of life gave values 5-13 mg/dl(300-780 PM). At 21 months of age, following two values of 16 mg/dl (1000 ,uM) and 15 mg/dl (900 PM), the infant was placed on a phenylalanine restricted diet. This patient was exclusively breastfed for the first 7 months of life. Stanford-Binet testing at age 3 showed her to be in the average range. Patient 3 was the first child of Anglo parents. The newborn screening result by Guthrie bacterial assay at 3 days of age was slightly greater than 4 mg/dl (240 PM). At 2 weeks of age the value by paper chromatography was 10 mg/dl (600 PM) and

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at 1 month of age a quantitative value by ion exchange column chromatography was 8.4 mg/dl (510 PM). She was considered to have MHP and a normal diet was continued. She was not seen again until age 21 months when she was evaluated in a followup study of MHP. Surprisingly, her plasma phenylalanine level was 18.5 mg/dl (1120 PM). After an overnight fast she was given a loading dose of 100 mg/kg of phenylalanine orally which produced a peak plasma phenylalanine concentration of 34.1 mg/dl (2060 PM) at 3 h post ingestion. She continued a normal diet and was lost to followup until 7 years of age when she was again seen. Her plasma phenylalanine level at that time was 13.9 mg/dl (845 PM). Psychological testing (WISC-R) at age 7 years showed average intellectual functioning. Patient 4 had a newborn screening phenylalanine level of 6 mgidl (360 PM) by Guthrie bacterial inhibition assay. Subsequent values over the next 11 months ranged from 5 to 9 mg/dl (300-540 PM). At 1 year of age blood phenylalanine was 15 mg/dl(920 PM). Subsequent levels were 9 mgdl(540 PM) (13 months), 13 mgdl(780 PM) (14 months), 18 mg/dl (1080 FM) (14.5 months), 14 mg/dl (840 PM) (15.5 months) and > 20 mg/dl (1200 PM) (16 months). The last level was obtained during an upper respiratory illness. Subsequent levels over the next 6 weeks were 16 mg/dl (1000 PM) and 11 mg/dl (660 PM). The patient was then placed on a protein restricted diet and the blood phenylalanine levels decreased to 5.4 mg/dl (330 PM) and 9.6 mg/dl (580 PM). 3. Discussion

These patients emphasize the need for long-term followup of even mildly elevated phenylalanine levels identified in newborn screening for PKU. The second patient we report took nearly 2 years to develop plasma levels of phenylalanine high enough to provoke dietary intervention. In the partially breastfed infant reported by Binder et al. [7], the blood phenylalanine level did not rise to above 20 mgdl (1200 PM) until between 4 and 7 months of age. In an abstract, Schwartz et al. [8] recently reported a male patient who was followed with phenylalanine levels that initially were 6-8 mg/dl (360-480 PM) and, at age 5 years, rose to levels of 10 mg/dl (600 FM) and 11 mg/dl (660 PM). This patient was then placed on a protein-restricted diet. We also have had experience with a number of other patients whose levels rose slowly during the first 2-6 months of life, eventually into the range for dietary intervention. The four cases we have reported took unusually long times to rise to these levels. Patients 1 and 2 were exclusively breastfed for the first 6-12 months of life. Human milk contains only 43 mg/dl of phenylalanine while the phenylalanine content of infant formulas is 59-73 mg/dl and of cow’s milk is 164 mg/dl. It is likely that the relatively low phenylalanine content of human milk contributed to the slow rise of the blood phenylalanine levels in these infants [9,10]. At the Hershey Medical Center monthly or bimonthly filter paper blood specimens for Guthrie bacterial inhibition assay are requested in any infant whose newborn screening phenylalanine level is above 4 mg/dl(240 PM). If repetitive values are below 8 mg/dl (480 FM) during the first year, annual specimens are requested

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thereafter. This latter practice may be particularly important in female children to assess the possibility of high risk in a future pregnancy [l 11. Genotyping the phenylalanine hydroxylase (PAH) gene as soon as possible after newborn screening identification of hyperphenylalaninemia could rapidly establish the definitive category for the affected infant and, in particular, could sort those infants with PKU from those with MHP, since there is a close association between the genotype and the biochemical phenotype [12]. The PCR-DGGE method with direct sequencing, developed by Guldberg et al. [ 13,141, seems particularly suited for this purpose. Since such genotyping is expensive and requires specific expertise, it is impractical for all newborn screening programs or individual medical centers to have this capability. An alternative would be to develop this capability in a single center or a very few centers in North America, either newborn screening programs or medical centers, to which specimens could be referred. References [I] [2] [3] [4] [5]

[6] [7] [8] [9] [IO]

[I I] [l2]

[I31 [14]

Therrel B, Panny SR, Davidson A et al. U.S. newborn screening system guidelines: statement of the Council of Regional Networks for Genetic Services. Screening 1992; I: 135. Ferriera P. Current Canadian newborn screening practices. Pediatr Med Q 1989; 3: I l I. Levy HL, Shih VE, Karolkewicz V et al. Persistent mild hyperphenylalaninemia in the untreated state. A prospective study. N Engl J Med 1971; 285: 424. Lang MJ, Koch R, Fishier K et al. Nonphenylketonuric hyperphenylalaninemia. Am J Dis Child 1989; 143: 1464. Hanley WB, Clarke JTR, Schoonheyt et al. What is a safe blood phenylalanine (phe) level for dietary treatment of PKU? A review of 31 patients with persistent benign hyperphenylalaninemia. Clin Invest Med 1989; 12: A34. Costello PM, Beasley MC, Tillotson SL et al. Intelligence in mild atypical phenylketonuria. Eur J Pediatr 1994; 153: 260. Binder J, Johnson CF. Saboe B et al. Delayed elevation of serum phenylalanine level in a breastfed child. Pediatrics 1979; 63: 334. Schwartz RC, Makaida C, Greenstein RM. Diagnosis of variant PKU in a child with the initial diagnosis of mild persistent hyperphenylalaninemia (MPH). Am J Hum Genet 1994; 55: A343. Miller ME, Plumeau P, Blakely E. Elevated phenylalanine concentrations in benign hyperphenylalaninemia from evaporated milk feedings. Clin Pediatr 1993; 32: 124. Greve LC, Wheeler MD, Green-Burgeson DK et al. Breastfeeding in the management of the newbornwith phenylketonuria: a practical approach to dietary therapy. J Am Diet Assoc 1994; 94: 305. Lenke RR, Levy HL. Maternal phenylketonuria and hyperphenylalaninemia: an international survey of the outcome of untreated and treated pregnancies. N Engl J Med 1980; 303: 1202. Guldberg P, Levy HL, Koch R et al. Mutation analysis in families with discordant phenotypes of phenylalanine hydroxylase deficiency. Inheritance and expression of the hyperphenylalaninemias. J Inher Metab Dis 1994; 17: 645. Guldberg P, Henriksen KF, Giittler F. Molecular analysis of phenylketonuria in Denmark: 99% of the mutations detected by denaturing gradient gel electrophoresis. Genomics 1993; 17: 141. Guldberg P, Roman0 V, Ceratto N et al. Mutational spectrum of phenylalanine hydroxylase defyciency in Sicily: implications for diagnosis of hyperphenylalaninemia in Southern Europe, Hum Mol Genet 1993; 2: 1703.