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PKU owing to Phe hydroxylase deficiency is one of the most common inborn errors of metabolism. Different recommendations do exist concerning the treatment (1–3). The Phe hydroxylase gene can be mapped to chromosome 12q22-q24.1 (4, 5). Up to now, >400 different mutant Phe hydroxylase alleles have been described (4).

On the basis of the serum Phe levels on a free nutrition, patients with HPA are classified as classic PKU (>1200 μM), mild PKU (600–1200 μM), or mild HPA (<600 μM) (2, 6). The classification limits are historic. There is international consensus that patients with Phe levels <360 μM on a free diet are not in need of a Phe-lowering dietary treatment, whereas patients with levels >600 μM are. Different recommendations exist for patients with Phe levels between 360 and 600 μM on a free nutrition.

In spite of early and adequate dietary treatment, patients with mild or classic PKU show a mild reduction of IQ (7–9), abnormalities of cerebral white matter (10, 11), and deficits of fine motor performances (12) and sustained and selective attention (13, 14), as well as impaired executive functions depending on (pre)frontal lobe functions (15, 16). The latter may be the consequence of a central dopamine depletion. In addition, neurologic deterioration was described in some adult patients who stopped their diet for more than 10 y (17).

The authors of a study from Great Britain (1, 18) in which the IQ was measured concluded that all patients with Phe levels >360 μM on a normal diet should be treated. In their study they included patients with Phe levels <900 μM. Most German patients with mild HPA are not treated, as a normal outcome was anticipated (2, 3).

In 1997 we performed a first systematic study and found normal intellectual, neurologic, and neuropsychological outcomes in 28 untreated subjects with mild HPA compared with healthy control individuals (3). However, in that study only 10 of 28 patients had Phe levels in the controversial range between 360 and 600 μM on a free nutrition. Our results were supported by findings of Levy et al.(19), who reported normal IQs in 13 4-y-old patients with mild HPA and who were comparable to the IQs of their unaffected siblings. In another study by Levy and Waisbren (20), eight women with mild HPA reached a normal mean IQ. In a study on maternal mild HPA, Levy et al.(21) found a normal median IQ of 105 in 37 of 86 women for whom IQ test results were available. Patients with Phe levels <400 μM had a higher median IQ (108) compared with patients with Phe levels above this level (100), but this difference was not significant (21). Waisbren et al.(22) found no intellectual deficits or abnormalities in personality in a group of 12 patients with untreated PKU (n = 6) and mild HPA (n = 6). Unfortunately no data were published on the socioeconomic status of the patients in these studies.

To overcome the limitation of our first study, we now investigated in a nationwide study adolescent and adult patients with mild HPA and Phe levels within the critical range between 360 and 600 μM on a normal nutrition. The hypothesis tested was that untreated subjects with mild HPA and Phe levels on a free nutrition between 360 and 600 μM are not at risk for developing intellectual, neurologic, and neuropsychological deficits.

METHODS

Subjects.

All departments of pediatrics of the 22 German University Hospitals were involved in this study. Four hospitals had no patients with mild HPA and Phe levels between 360 and 600 μM on a free diet, whereas at six hospitals these patients were treated with a Phe-restricted diet. At 12 hospitals there were altogether 40 patients with untreated mild HPA >10 y of age. Thirty-one of these 40 patients were willing and able to take part in our study (Table 1), five patients were not available, and four patients did not consent to participate because they expected no benefit for themselves. These patients did not significantly differ from the participating subjects in socioeconomic status and school career (information by telephone).

Table 1 Description of study groups
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Healthy control subjects (friends of patients) were selected according to age, sex, and socioeconomic status. Informed consent was obtained from all patients and healthy control subjects and their parents. The study was approved by the Ethics Committee of the University of Münster. Patients with mild HPA were defined by serum Phe levels persistently between 360 and 600 μM in the neonatal screening test and sporadic tests throughout life, as well as four determinations at 2-wk intervals during the study (mean study levels;Table 2). Serum Phe levels during the study were measured by amino acid analyzer (HPLC). Blood samples were taken 4–5 h after the last meal. For 29 patients, Phe levels of the neonatal screening test (first quantitative measurement within 1 wk after the Guthrie test, n = 22) or Guthrie bacterial inhibition assay for Phe (n = 29) were available.

Table 2 Serum phenylalanine levels
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One older patient included in the sample was born before the general screening program was introduced, and for one patient the result of the screening test was not available. Four patients were characterized by a Blascovics Phe load at the age of 4–6 mo (23). A mean lifetime serum Phe level was not computed because Phe checks were performed only sporadically. Socioeconomic status was defined with the help of the variables family income, academic qualification, and job of the father as described earlier (3). Concerning school and job career, we obtained informations of altogether 31 patients and their siblings by asking their parents.

Mutation analysis.

All 13 exons and flanking intronic sequences of the Phe hydroxylase gene from 30 non-PKU HPA subjects were amplified by PCR and scanned for the presence of mutations by denaturant grading gel electrophoresis according to previously described procedures (24). All samples displaying an altered electrophoretic band pattern were subjected to direct sequencing. In general, genotyping may help to categorize the patients as mild HPA at an early stage of life.

Assessment procedures.

The assessment program consisted of tests that had previously revealed significant deficits in patients with classic or mild PKU who were treated early (7–14). These tests had already been used in our recent study on patients with mild HPA (3), where they are described in detail. Patients and controls were examined for clinical-neurologic outcome, IQ [German version of Wechsler intelligence scale for adults—revised and Wechsler intelligence scale for children—revised;(25, 26)], school performance and job career, selective attention [Color Word Interference Task = Stroop task;(27)], and sustained attention [dot pattern exercise from the Sonneville Visual Attention Task;(28)], as well as for their fine motor performances [Motor Performance Task;(29)]. In addition, cranial MRI and 1H MR spectroscopy were obtained in 10 patients using a superconducting 1.5-T magnet (Magneton, Siemens) using spin-echo pulse sequences. T1-weighted and proton density-weighted images [transmission time (TR), 2500; echo time (TE) 15/90] were acquired in the axial plane. In addition coronal T2-weighted images (TR 600, TE 1.5) were obtained. Brain Phe concentrations were determined by localized 1H spectroscopy applying the STEAM technique on a 1.5-T MR unit (Siemens, Germany) in 10 patients. Spectra were recorded from periventricular occipital brain as described previously [TR/TE, 1500/20 ms; 512 acquisitions; 36-mL voxels;(30)]. The 10 patients had mean study Phe levels of 440 ± 110 μM.

Data analysis.

Nonparametric tests (Wilcoxon test, correlation according to Spearman, χ2 test) were used for statistical evaluation.

RESULTS

Genotypes and corresponding biochemical phenotypes.

The present investigation supports previous suggestions that mild HPA is caused in most cases by compound heterozygosity for a PKU mutation and mild HPA mutation (5) (Table 3).

Table 3 Genotypes and serum phenylalanine levels of 31 patients
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Clinical-neurologic outcome.

Clinical-neurologic examination revealed mild rest and intention tremor in one subject with mild HPA and clumsy diadochokinesis in a control subject. Physical development was normal in all subjects with mild HPA and in all healthy control subjects.

Intelligence.

The 31 subjects with mild HPA attained a mean IQ within the normal range. Verbal IQ and performance IQ did not significantly differ (Table 4).

Table 4 Intelligence score
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School performance.

The 31 patients, their 31 siblings, and the 31 healthy control subjects did not significantly differ with regard to age at start of schooling, types of schools, and repetition of classes (Table 5).

Table 5 School performance
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Job career.

Patients and siblings, as well as healthy control subjects, did not significantly differ with respect to their jobs (Table 6).

Table 6 Job career
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Sustained attention.

Levels of performance as well as the stability of performance on the dot pattern exercises of Sonneville Visual Attention Task were not significantly different for subjects with mild HPA and healthy control subjects (Table 7).

Table 7 Sustained attention dot pattern exercise
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Selective attention.

The Color Word Interference Task, which depends on frontal lobe functions, revealed no significant difference between subjects with mild HPA and control subjects (Table 8).

Table 8 Selective attention (Color Word Interference Task, Stroop task)
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Fine motor performance.

The Motor Performance Task revealed no significant fine motor deficits in patients with mild HPA (Table 9).

Table 9 Motor performance task—dominant right hand
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MRI.

The blinded evaluation of patients' scans revealed no abnormalities in any case, especially no alterations of cerebral white matter.

1H MR spectroscopy.

In all patients brain Phe concentrations were below the limit of detection (<200 μM).

Correlational findings.

The Phe levels of the screening test and the mean study levels were significantly correlated (r = 0.57, p < 0.001). Correlational analysis according to Spearman revealed no significant relation between serum Pe levels (screening test and mean study level) and results of psychological measures.

DISCUSSION

Data presented confirm the results of our recent study on patients with mild HPA (3). Adolescent and adult patients with mild HPA and serum Phe levels in the disputed range of 360 to 600 μM seem to be not at risk for developing significant intellectual, neurologic, and neuropsychologic deficits or abnormalities of cerebral white matter, as described for patients with mild or classic PKU (7–14). Because of the limited accuracy of measurements, Phe levels should be taken as an approximation but not as absolute limits in every instance.

Our results are supported by findings of Levy et al.(19), Levy and Waisbren (20), Levy et al.(21), and Waisbren et al.(22). The discrepancy between the IQ data of the present study and the study from Great Britain are possibly related to the inclusion of patients with Phe levels between 600 and 900 μM in the British sample (1).

In the present study, as in our previous study on mild HPA (3), results of different tests were not significantly correlated with Phe levels (neonatal screening as well as mean study levels).

In conclusion, in the absence of any effect on clinical outcome, treatment is unlikely to be of benefit to subjects with mild HPA and serum Phe levels <600 μM on free nutrition. In addition brain Phe levels <200 μM seem to allow a normal cognitive development. Our results support the recommendations of the German Working Group for Metabolic Diseases from 1999, supposing that only patients with HPA caused by phenylalanine hydroxylase (PAH) deficiency and serum Phe levels of ≥600 μM on a free nutrition must be treated (2). Because of a possible late-onset PKU, untreated patients should be monitored very carefully at least during the first year of life (4). Further studies are needed to clarify whether 600 μM is the exact limit for this treatment decision. A very burdensome, stigmatizing, and expensive dietary treatment should be avoided.

Nevertheless, problems of maternal PKU should be intensively discussed with women with mild HPA and their relatives, as their elevated serum Phe levels during pregnancy may be associated with damage to the fetus (4, 21).