School of Medicine

Department of Genetics

Phenylketonuria in Louisiana

Lindsay Burrage
Hans C. Andersson

Phenylketonuria (PKU), a classic example of an inborn error of metabolism, was first described by Asbjörn Fölling in 1934 when he discovered phenylpyruvic acid in the urine of two mentally retarded siblings. Following this discovery, he analyzed the urine of over 400 mentally retarded patients and discovered eight other patients whose urine contained abnormal quantities of phenylpyruvic acid. Each of the patients had a similar clinical presentation or phenotype with features including mental retardation, a "mousy" odor, and dermatitis. Further studies indicated that the disorder is inherited as an autosomal recessive trait.

The exact metabolic defect in PKU was not elucidated until 1947. After administering phenylalanine (PHE) to a group consisting of both normal controls and affected patients. Jervis noticed that there was an increase in the concentration of tyrosine (TYR) in the blood of the controls but not in PKU patients, and he concluded that the hydroxylation of PHE to TYR was deficient in PKU patients. In 1953, Jervis first demonstrated deficient activity of phenylalanine hydroxylase (PAH) (EC in the liver of PKU-affected patients. PAH is primarily active only in the liver, and enzymatic activity can only be measured in this tissue.

The classical and variant forms of PKU are caused by a deficiency of the hepatic phenylalanine hydroxylase (PAH). PAH is a homotetramer that oxidizes PHE to form TYR This oxidation is a critical step in the pathway in the conversion of PHE to carbon dioxide and water as well for the formation of TYR, which is an essential amino acid in the diet of PKU patients. The gene which codes for the PAH enzyme is mapped to chromosome 12q22-q24.1. The PAH gene is nearly 90 kb in length and consists of 13 exons. About 400 mutations in the PAH gene have been discovered, and although some of these mutations appear to have no affect on the enzyme's function, most have been associated with either classical or variant PKU.

The phenotypic heterogeneity in classical and variant PKU is a result of the vast number of PAH mutations that are responsible for the disorders. For the most part, particular mutations can be correlated with specific phenotypes. For example, "severe" mutations result in higher plasma PHE levels (classical PKU) than do "mild mutations". Compound heterozygosity for one of the "severe" mutations and one of the "mild" mutations results in intermediate plasma PHE levels. Furthermore, patients that are homozygous for a "mild" mutation or compound heterozygotes for two different "mild" mutations generally have plasma PHE levels that are characteristic of variant rather than classical PKU. In contrast, there are a few cases in which the mutations cannot be correlated with the patient's Clinical Phenotype.

Clinical Phenotype
With the advent of newborn screening methods, hyperphenylalaninemia patients are generally diagnosed soon after birth. The blood concentrations of PHE in untreated classical PKU patients with normal protein intake is usually over 1200 mcmol/L (19.8 mg/dL) phenotype . These patients generally have less than 5% of the normal enzyme activity. Blood PHE concentrations in untreated patients that are slightly elevated compared to normal (greater than 151 mcmol/L but less than 1200 mcmol/L or 2.5- 19.8 mg/dL) are indicative of a milder form termed variant PKU or may be due to transient hyperphenylalaninemia of the newborn. If the slight elevation of PHE disappears within the first three months, the patient is presumed to have transient hyperphenylalaninemia. Transient hyperphenylalaninemia is believed to be due to an immature PAH system, which is developmentally induced during infancy. Most patients with variant PKU do not require treatment unless the plasma PHE rises above 484-605 mcM (8-10 mg/dL). All patients with elevated plasma PHE must be tested for non-PAH forms of the disease since plasma PHE levels do not suffice to distinguish these types.

Untreated PKU patients develop moderate to profound mental retardation and have a characteristic "mousy" odor, fair complexion, abnormal gait and stance, and dermatitis. The impaired brain functioning in PKU patients is felt to be due to the excess of PHE and PHE-metabolites in the brain, but the exact pathophysiology remains unclear. The abnormal levels of PHE prevent the normal transport of other amino acids, particularly TYR, across the blood-brain barrier . As a result, neurotransmitter synthesis and protein synthesis in the brain is disrupted. In addition, the structure of the brain cells is abnormal, and myelination is defective.

In 1954, Bickel demonstrated that dietary restriction of PHE effectively reduces plasma PHE and urine phenylpyruvate levels. In addition, the mental development of PKU patients on the PHE-restricted diet was improved. Further studies revealed that the PHE-restricted diet prevents mental retardation and other phenotypic effects of the disorder. As a result, dietary therapy has become the standard treatment for PKU. It is recommended that PKU patients maintain plasma PHE levels of between 121-484 mcM or 2-8 mg/dL (normal values are 30-121 mcM or 0.5-2.0 mg/dL) throughout childhood, adolescence, and even adulthood. Various low-PHE products are commercially available for PKU patients particularly synthetic, low-PHE formulas. These formulas are supplemented with free amino acids, vitamins, and minerals, but unfortunately, the poor taste and odor of the formulas contribute to problems with dietary compliance.

Adequately treated PKU patients do not develop mental retardation. Some studies suggest that the IQ of continuously treated PKU patients may be slightly decreased as compared to control subjects. Other studies indicate that strict dietary adherence throughout childhood and adolescence may eliminate most of the differences in test scores between PKU patients and control subjects. According to some studies, the only area where the test scores of PKU patients fall significantly below those of control subjects is mathematics. In contrast, the results of a more recent study indicate that the spatial intelligence of early and continuously treated patients is poor even though verbal intelligence and arithmetic skills are normal. Overall, the I.Q. of PKU patients appears to be significantly affected by delays in the initiation of the low-PHE diet, prolonged exposure to PHE levels above the treatment range, and five or more months of exposure to PHE levels below the target range during the first two years.

Newborn Screening for the Hyperphenylalaninemias
Since early dietary therapy is necessary to achieve optimal clinical outcome, hyperphenylalaninemia patients must be identified as soon as possible after birth. In 1961, Dr. Robert Guthrie developed a method for the early detection of newborn patients. His assay utilized the inhibitory effect of a phenylalanine analogue on the growth of the bacteria Bacillus subtilis. In the presence of PHE, the inhibitory effect is overcome, and the area of growth can be used to measure the quantity of PHE in blood. Blood for the assay can readily be obtained from a filter paper blood spot taken 48 hours after birth. In 1965, a fluorometric method, which also utilizes filter paper blood spots, was developed. The fluorometric method relies on the fact that PHE forms a fluorescent substance when it is heated with ninhydrin and L-leucyl-L-alanine. The amount of fluorescence indicates the quantity of PHE present in the sample. This method has replaced the bacterial inhibition assay in many states including Louisiana. The threshold level of PHE for positive samples is 121-242 mcM or 2-4 mg/dL, the normal upper limit of plasma PHE, and all positive samples are referred for amino acid analysis for a definitive diagnosis.

Maternal PKU
The current dietary recommendations for PKU patients stress dietary therapy for life, especially for affected women because high plasma PHE acts as a fetal teratogen. Nearly 80% of fetuses exposed to high levels of PHE during development have microcephaly, mental retardation, and growth retardation. A smaller percentage are born with heart defects and other congenital malformations including dysmorphic facial features. The level of fetal abnormalities correlates with the maternal plasma PHE levels, and as a result, women who maintain low plasma PHE (below 605 mcM or 10 mg/dL) have a lower risk of having a child with maternal PKU. Women with PKU must maintain proper dietary control prior to conception since high levels of PHE have the most severe detrimental effects in the first trimester

PKU In Louisiana
Between 1985 and 1999, 67 (.0067%) of approximately 1,000,000 newborns in Louisiana were diagnosed with a form of hyperphenylalaninemia. Sixty-six of the patients were diagnosed with either classical PKU or variant PKU, and the overall incidence of PKU (classical and variant) in Louisiana during the study period was 1:16,000. One patient was diagnosed with 6-pyruvoyl tetrahydropterin synthase (6-PTS) deficiency, a non-PAH form of hyperphenylalaninemia during the study period. No patients were diagnosed with transient hyperphenylalaninemia of the newborn. All 67 patients were identified by the Louisiana newborn screening program and cared for by the Tulane Medical School Human Genetics Program. Of the 66 PKU patients born in Louisiana, 60 (91%) were white, and six (9%) were non-white. Five of the non-white patients were African-American, and one had a mixed racial background (white and African-American). No Asian PKU patients were born during the study period. 

Tulane Medical School Human Genetics Program: