Flemming Güttler

A European multicenter study of phenylalanine hydroxylase deficiency: Classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%-23% (P<.0001), suggesting that differences in methods used for mutation detection or phenotype classification may account for a considerable proportion of genotype-phenotype inconsistencies. Our data indicate that the PAH-mutation genotype is the main determinant of metabolic phenotype in most patients with PAH deficiency. In the present study, the classification of 105 PAH mutations may allow the prediction of the biochemical phenotype in >10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.

Molecular basis of phenotypic heterogeneity in phenylketonuria.

BACKGROUND Phenylketonuria is a metabolic disorder that results from a deficiency of the hepatic enzyme phenylalanine hydroxylase. Its clinical phenotype varies widely, and to date more than 10 mutations in the phenylalanine hydroxylase gene have been identified in persons with the disorder. We attempted to relate the clinical phenotype of patients to their genotype. METHODS We studied 258 patients with phenylketonuria from Denmark and Germany for the presence of eight mutations previously found in patients from these countries. The in vitro activity of the enzymes associated with these mutations was determined by expression analysis in heterologous mammalian cells. The level of activity was then used to predict the in vivo level of phenylalanine hydroxylase activity in patients with various combinations of mutant phenylalanine hydroxylase alleles. RESULTS The eight mutations involved 64 percent of all mutant phenylalanine hydroxylase alleles in the patients. Expression analysis showed that these mutant enzymes produced from 0 to 50 percent of normal enzyme activity. The predicted level of phenylalanine hydroxylase activity correlated strongly with the pretreatment serum level of phenylalanine (r = 0.91, P less than 0.001 in the Danish patients and r = 0.74, P less than 0.001 in the German patients), phenylalanine tolerance in the Danish patients (r = 0.84, P less than 0.001), and the serum phenylalanine level measured after standardized oral protein loading in the German patients (r = 0.84, P less than 0.001). CONCLUSIONS Our results strongly support the hypothesis that there is a molecular basis for phenotypic heterogeneity in phenylketonuria. The establishment of genotype will therefore aid in the prediction of biochemical and clinical phenotypes in patients with this disease.

Normal Clinical Outcome in Untreated Subjects with Mild Hyperphenylalaninemia

There is international consensus that patients with phenylalanine (Phe) levels <360 μM on a free diet do not need Phe-lowering dietary treatment whereas patients with levels >600 μM do. Clinical outcome of patients showing Phe levels between 360 and 600 μM in serum on a free nutrition has so far only been assessed in a small number of cases. Therefore, different recommendations exist for patients with mild hyperphenylalaninemia. We investigated in a nationwide study 31 adolescent and adult patients who persistently displayed serum Phe levels between 360 and 600 μM on a normal nutrition with a corresponding genotype. Because of limited accuracy of measurements, Phe levels should be looked on as an approximation, but not as an absolute limit in every instance. In addition to serum Phe levels, the assessment program consisted of comprehensive psychological testing, magnetic resonance imaging of the head, 1H magnetic resonance spectroscopy, and genotyping. We found a normal intellectual (intelligence quotient, 103 ± 15; range, 79–138) and educational (school performance and job career) outcome in these subjects as compared with healthy control subjects (intelligence quotient, 104 ± 11; range, 80–135). Magnetic resonance imaging revealed no changes of cerebral white matter in any patient, and 1H magnetic resonance spectroscopy revealed brain Phe levels below the limit of detection (<200 μM). In the absence of any demonstrable effect, dietary treatment is unlikely to be of value in patients with mild hyperphenylalaninemia and serum Phe levels <600 μM on a free nutrition, and should no longer be recommended. Because of a possible late-onset phenylketonuria, Phe levels of untreated patients should be monitored carefully at least during the first year of life. Nevertheless, problems of maternal phenylketonuria should still be taken into account.

Biopterin responsive phenylalanine hydroxylase deficiency

Purpose: Phenylketonuria (PKU) is an autosomal recessive disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. There have been more than 400 mutations identified in the PAH gene leading to variable degrees of deficiency in PAH activity, and consequently a wide spectrum of clinical severity. A pilot study was undertaken to examine the response to 6-R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) in patients with atypical and classical PKU.Methods: PAH gene mutation analysis was performed using denaturing gradient gel electrophoresis and gene sequencing. Patients with classical, atypical, or mild PKU were orally given BH4 10 mg/kg. Blood phenylalanine and tyrosine levels were determined using tandem MS/MS at 0 hours, 4 hours, 8 hours, and 24 hours intervals.Results: Thirty-six patients were given a single oral dose of 10 mg/kg of BH4. Twenty one patients (58.33%) responded with a decrease in blood phenylalanine level. Of the patients that responded, 12 were classical, 7 atypical, and 2 mild. The mean decline in blood phenylalanine at 24 hours was > 30% of baseline. There were 15 patients who did not respond to the BH4 challenge, 14 of those had classical and one had atypical PKU. Mapping the mutations that responded to BH4 on the PAH enzyme showed that mutations were in the catalytic, regulatory, oligomerization, and BH4 binding domains. Five patients responding to BH4 had mutations not previously identified.Conclusion: The data presented suggest higher than anticipated number of PKU mutations respond to BH4, and such mutations are on all the domains of PAH.

Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria

SummaryEight polymorphic restriction enzyme sites at the phenylalanine hydroxylase (PAH) locus were analyzed from the parental chromosomes in 33 Danish nuclear families with at least one phenylketonuric (PKU) child. Determination of haplotypes of 66 normal chromosomes and 66 chromosomes bearing mutant allele (S) demonstrated that there are at least two haplotypes which occur predominantly on PKU chromosomes and rarely otherwise. Overall, the relative frequencies of the various haplotypes are significantly different on PKU-and normal-allele bearing chromosomes, even though there is no predominantly occurring unique haplotype which can characterize the PKU chromosomes. In addition, no significant association (linkage disequilibrium) between any single polymorphic site and the mutant allele (s) was observed. The results suggest that either the phenylketonuric mutation was very ancient so that the polymorphic sites and the mutation have reached linkage equilibrium or the mutant allele (s) are the results of multiple mutations in the phenylalanine hydroxylase gene in man. Furthermore, a crude relationship between standardized linkage disequilibria and physical map distances of the polymorphic sites indicates that there is no apparent recombination hot-spot in the human phenylalanine hydroxylase gene, since the recombination rate within the locus apears to be uniform and likely to be occurring at a rate similar to that within the HLA gene cluster. The limitations of this later analysis are discussed in view of the sampling errors of disequilibrium measure used, and the potential untility of the PAH haplotypes for prenatal diagnosis and detection of PKU carriers is established.

Double blind placebo control trial of large neutral amino acids in treatment of PKU: Effect on blood phenylalanine

SummaryLarge neutral amino acids (LNAA) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In an open-label study using LNAA, a surprising decline of blood Phe concentration was found in patients with PKU in metabolic treatment centres in Russia, the Ukraine, and the United States. To validate the data obtained from this trial, a short-term double-blind placebo control study was done using LNAA in patients with PKU, with the participation of three additional metabolic centres – Milan, Padua and Rio de Janeiro. The results of the short trial showed significant lowering of blood Phe concentration by an average of 39% from baseline. The data from the double-blind placebo control are encouraging, establishing proof of principle of the role of orally administered LNAA in lowering blood Phe concentrations in patients with PKU. Long-term studies will be needed to validate the acceptability, efficacy and safety of such treatment.

Blood-brain phenylalanine relationships in persons with phenylketonuria.

Objectives. Clinicians caring for persons with phenylketonuria (PKU) have been perplexed by the occasional normal individual with the classical biochemical profile consistent with the diagnosis of PKU. Usually untreated subjects with the biochemical profile of blood phenylalanine (Phe) levels >1200 μmol/L are severely mentally retarded and may have neurological findings. Preliminary reports have recently appeared suggesting that low brain Phe levels, in comparison with elevated blood Phe levels, account for the occurrence of these occasional unaffected individuals with the biochemical profile consistent with PKU. Method. Magnetic resonance imaging/magnetic resonance spectroscopy was used to measure brain Phe content compared with simultaneously obtained blood Phe levels determined on the amino acid analyzer. This comparison was obtained in 5 normal non-PKU persons, 4 carriers of the gene causing PKU, and in 29 individuals with the proven form of the disorder. Results. Blood–brain measurements in 5 normal persons ranged from .051 to .081 mmol/L, with a mean of .058 mmol/L. Their simultaneously measured brain levels of Phe ranged from .002 to .15 mmol/L, with a mean of .09 mmol/L. Similar measurements were obtained in 4 carriers of the gene causing PKU. Their blood levels varied between .068 and .109 mmol/L, with a mean of .091 mmol/L and simultaneously obtained brain levels of Phe varied between .06 and .21 mmol/L, with a mean of .11 mmol/L. Twenty subjects with a mean IQ of 104 exhibited a mean blood level of 1.428 mmol/L and a simultaneous mean brain level of .23 mmol/L, whereas 9 persons with a mean IQ of 98.7 exhibited a mean blood Phe level of 1.424 and a mean brain Phe level of .64 mmol/L. The correlation between blood and brain levels was not significant. Conclusion. In usual cases, intellectually normal persons who have never been treated but who have a biochemical profile consistent with classical PKU exhibit lower brain levels of Phe. Such individuals are exceptional and may not need the vigorous restriction of their blood Phe levels that is required by the majority of persons with PKU.

The international collaborative study of maternal phenylketonuria: status report 1994

Neonatal screening for phenylketonuria (PKU) has created a problem as females with PKU are reaching child‐bearing age. Surveys have revealed that maternal phenylalanine blood concentrations greater than 1200 μmol/l are associated with fetal microcephaly, congenital heart defects and intrauterine growth retardation. It is estimated that as many as 3000 hyperphenylalaninemic females may be at risk of producing these fetal abnormalities. To examine this problem, the international maternal PKU collaborative study was developed to evaluate the efficacy of a phenylalanine‐restricted diet in reducing fetal morbidity. Preliminary findings have indicated that phenylalanine restriction should begin before conception for females with PKU planning a pregnancy. Dietary control should maintain maternal blood phenylalanine levels between 120 and 360 μmol/l and should provide adequate energy, protein, vitamin and mineral intake. Pregnant hyperphenylalaninemic females who achieved metabolic control after conception or by the 10th week of pregnancy had a better offspring outcome than anticipated. The results of 402 pregnancies are reviewed.

Increased neurotransmitter biosynthesis in phenylketonuria induced by phenylalanine restriction or by supplementation of unrestricted diet with large amounts of tyrosine

Seven phenylketonuria (PKU) patients aged 15–24 years were allowed unrestricted diet for 3 weeks. Three of these patients performed well on unrestricted diet according to visual reaction time variability (RTv 50–100 ms) and did not show significant changes when returning to the phenylalanine-restricted diet (RTv 70–100 ms). Neither did the concentrations of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid (CSF) change significantly. Four of the patients, however, performed rather poorly (RTv 120–220 ms) on unrestricted diet and improved significantly (P<0.03) when the diet was restored (RTv 70–150 ms). The improvements were accompanied by significant (P<0.01 and P<0.02) increases (mean 52% and 109%) in CSF levels of HVA and 5-HIAA. Five PKU patients aged 15–23 years were allowed unrestricted diet or unrestricted diet supplemented with various amounts of tyrosine (106–194 mg/kg per 24 h). Two of these patients performed very well on unrestricted diet (RTv 60 ms) and showed little change when the unrestricted diet was supplemented with tyrosine (RTv 70 ms and 80 ms). The three other patients, who performed rather poorly (RTv 120–220 ms), improved significantly (P<0.03) when the unrestricted diet was supplemented with tyrosine (RTv 70–140 ms). HVA in CSF increased significantly (P<0.01) with the tyrosine supplement when the amount exceeded a threshold of approximately 80 mg/kg per 24 h. The simultaneous increase in CSF level of 5-HIAA showed a positive correlation (r=0.90; P<0.02) with the increase in HVA concentration suggesting a functional interrelation between the dopaminergic and the serotoninergic nervous systems.

Large neutral amino acids in the treatment of phenylketonuria (PKU)

SummaryLarge neutral amino acids (LNAAs) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In earlier studies on mice with PKU (ENU2/ENU2), LNAAs were given and a surprising decline in blood Phe concentrations was observed. The formula used in the mouse experiment (PreKUnil) lacked lysine. Therefore, a new formulation of LNAAs (NeoPhe) was developed, introducing changes in the concentration of some amino acids and adding lysine, so that such a mixture could be used in humans. The new formula was found to be effective in reducing blood Phe concentration in mice by about 50% of the elevated levels. Patients with PKU were given LNAAs and blood Phe concentrations were determined in an open-label study. Three centres—in Russia, the Ukraine and the USA—took part in the study. NeoPhe was given at 0.5 g/kg per day in three divided doses to eight subjects with PKU and at 1.0 g/kg per day to three patients, for one week. The NeoPhe resulted in decrease of elevated blood Phe by 50% in both groups. The preliminary data from this study are encouraging and a double blind placebo-controlled trial will be required to show long-term efficacy and tolerance of LNAAs in the treatment of PKU.