Phenylketonuria

Introduction and Facts

Phenylketonuria (PKU) is a congenital amino acid metabolism disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene located on chromosome 12q23.2. The decrease in PAH activity in PKU and HPA was caused by mutations in the PAH gene which resulted in the formation of non-functional PAH enzymes.

Phenylketonuria was first reported by a Norwegian doctor, Asbjorn Folling. In 1934, a mother of two children who suffered from intellectual disabilities was treated and asked if the strange smell in her children's urine was related to their intellectual impairment. The children's urine was tested for substances contained in it, including ketones.

The prevalence of phenylketonuria varies worldwide; in the Caucasian race between 1: 10,000 to 1: 15,000. The highest incidence is in Turkey (1 : 2,600) and in Iran (1 : 4698), due to the high number of consanguineous marriages (family relations) in these populations. The lowest incidence was found in the population in Japan (1 : 125,000) and in Finland (1 : 200,000).

Pathophysiology

Phenylalanine can enter the brain via the neutral aminoacid carrier-Laminoacid transporter1 (LAT1). Elevated levels of phenylalanine in the brain can impair neurophysiological function through several mechanisms. From the results of radiological imaging found white matter lesions associated with reduced myelin formation, although a definite causative relationship has not been found between dysmyelination and neurophysiological disorders.

Other neutral amino acids, namely tyrosine, which is a precursor of dopamine and norepinephrine and tryptophan which is a precursor of serotonin, also enter the brain via LAT1 carriers. High blood levels of phenylalanine can inhibit LAT1 and other neutral amino acids from entering the brain, increasing the risk of neurotransmitter and protein synthesis dysfunction. Other mechanisms of brain damage due to hyperphenylalaninaemia are reduced pyruvate kinase activity, impaired glutamatergic neurotransmission and reduced activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The clinical significance of reduced dopamine, catecholamines, and serotonin in the brain in PKU patients is not clearly understood. Among these neurotransmitters, dopamine has been studied extensively. Decreased dopamine can cause problems in prefrontal neurons, which have a higher dopamine turnover than other neurons in the brain. Patients with untreated PKU may develop chorea, tremor, and dystonia, probably due to a deficiency of dopamine in the basal ganglia. Cerebral serotonin deficiency may explain the increased anxiety and depression in patients with PKU.

Research studying oxidative stress in animal models of PKU found that lipid peroxidation as measured by malondialdehyde (MDA) was significantly higher in the brain and erythrocytes of PKU animals than in controls. Glutathione disulfide levels were also significantly reduced in the blood and brain of PKU animals.

Clinical Symptoms and Complications

At birth, the typical PKU infant is believed to have a normal nervous system. The disease appears later, only after long exposure of the nervous system to phenylalanine (PA), because the homozygous infant lacks the means of protecting the nervous system. However, if the mother is homozygous with high PA levels in the blood during pregnancy, the CNS is damaged in utero and the heterozygous infant is mentally defective from birth.

The damage done to the brain if PKU is untreated during the first months of life is not reversible. It is critical to control the diet of infants with PKU very carefully so that the brain has an opportunity to develop normally. Affected children who are detected at birth and treated are much less likely to develop neurological problems or have seizures and intellectual disability (though such clinical disorders are still possible.

In the classic form of PKU, the impairment of psychomotor development can usually be recognized in the latter part of the first year, when expected performance lags. By 5 to 6 years in an untreated child, when the IQ can be estimated, it is usually less than 20, occasionally 20 to 50, and exceptionally above 50. Hyperactivity, aggressivity, self-injurious behavior—including severe injury to the eyes, clumsy gait, fine tremor of the hands, poor coordination, odd posturings, repetitious digital mannerisms and other so-called rhythmias, and slight corticospinal tract signs stand out as the main clinical manifestations. Athetosis, dystonia, and frank cerebellar ataxia have been described but must be rare. Also, seizures occur in a small minority of severely affected patients (abnormal EEG finding), taking the form at first of flexor spasms and later of absence and grand mal attacks. The majority of PKU patients are blue-eyed and fair in skin and hair color, and their skin is rough and dry and subject to eczema. A musty or mousy body odor (because of phenylacetic acid).

Diagnosis

Neonate Screening

Phenylketonuria was identified through national neonatal screening. The first efficient test to detect hyperphenylalaninemia was the bacterial inhibition test developed by Robert Guthrie. The basis of this test is that Bacillus subtilis requires phenylalanine for its growth. The Guthrie test is very useful for mass screening of dried blood spot (DBS) samples using standardized filter paper (Guthrie Card) and sent to reference laboratories in envelopes. Tandem massspectrometry (TMS) was developed as a method to rapidly determine amino acid levels quantitatively in small volumes of blood/plasma samples. This method gives a smaller false positive result by measuring the levels of phenylalanine and tyrosine and gives the results of the phenylalanine/tyrosine ratio.

BH4 Loading Test

The BH4 loading test is used to differentiate phenylalanine elevations from PAH deficiency or BH4 deficiency (enzyme defects in biosynthesis or regeneration of BH4 cofactor). This test is useful in the early detection of BH4 deficiency and in the detection of PKU patients who are responsive to BH4 administration.

Cerebrospinal Fluid Examination

BH4 deficiency affects the synthesis of catecholamines, serotonin, and nitric oxide in the central nervous system and measurement of these metabolites in the cerebrospinal fluid is important for diagnosing the degree of BH4 deficiency. Assessment of not only absolute levels of 5-hydroxyindolacetic acid and homovanillic acid in cerebrospinal fluid, the ratio of neurotransmitters is also important in providing diagnostic information related to the severity and outcome of BH4 deficiency.

Treatment and Management

When diagnosed early, classic PKU can be treated with lifelong dietary therapy focused on maintaining low Phe levels and adequate Tyr intake. These dietary interventions are generally effective in preventing severe cognitive impairment due to high Phe levels. However, dietary therapy for PKU has been associated with deficiencies in selenium, copper, magnesium, and zinc.

The management of PKU is very complex, and dietary non-compliance often increases during adolescence and young adulthood, especially due to social issues. Besides a low Phe diet, promising drugs are being developed for PKU. Oral sapropterin dihydrochloride (KUVAN), a synthetic form of BH4, can help lower Phe levels in some PKU patients. It is currently not possible to predict which PKU patients will respond to BH4, but a 30-day trial can be conducted to determine this. Encouragingly, treatment with sapropterin dihydrochloride improves brain function in some PKU patients.

Enzyme replacement therapy for PKU has not been possible because PAH is unstable. The FDA has recently approved an enzyme substitution therapy for PKU. In this approach, a "substitute" enzyme is given to PKU patients that can lower Phe levels. This substitute enzyme is phenylalanine ammonia lyase coated with PEG (called pegvaliase) which can degrade Phe. Pegvaliase is approved only for adult patients with uncontrolled phenylalanine levels. It is still unclear whether pegvaliase will allow a less strict diet or provide long-term benefits in preventing or reversing cognitive impairment.

Women of childbearing age with PKU should receive counseling on the benefits of strict dietary therapy before and during pregnancy. Elevated maternal Phe levels during pregnancy can cause fetal brain damage and congenital heart disease. Adverse fetal effects due to high Phe levels during pregnancy can occur regardless of whether the fetus has a PKU variant.

The treatment of IEMs (Inborn Errors of Metabolism) has traditionally relied on treating the nutritional and/or metabolic environment, such as enzyme replacement therapy, rather than addressing the genetic disorder itself. Rapid advances in gene therapy and its safety may one day become possible. As of 2018, phase 1 gene therapy trials for PKU remain in the planning stages.

References:

1. Kurniawan LB. Patogenesis, skrining, diagnosis, dan penatalaksanaan phenylketonuria. Cermin Dunia Kedokteran 2015;232:42(9):668-73
2. BAHAN AJAR IV. Phenylketonurias (phenylalanine hydroxylase deficiency). [Internet]. [Cited: 27/8/2021]. Available from:https://med.unhas.ac.id/kedokteran/wp-content/uploads/2016/09/Bahan-Ajar-4_Fenilketonuria.pdf
3. Stone WL, Basit H, Los E. Phenylketonuria. National Library of Medicine [Internet]. 2023. Available from: https://www.ncbi.nlm.nih.gov/books/NBK535378/