I.  Definition

Phenylketonuria (commonly known as PKU) is one of inborn error of protein metabolism diseases that results from an impaired ability to metabolize the essential amino acid phenylalanine (Phe).1,2 PKU is an inherited disorder autosomal recessive and it is the most common of inborn error of amino acid metabolism. Deficiency of the enzyme phenylalanine hydroxylase (PAH) causes the accumulation of phenylalanine in body fluids, disturbs the normal brain development, and leads to mental retardation.1 The excess phenylalanine then convert to phenylketones that will appear in the urine, so it is commonly called by phenylketonuria.

II.    History

Phenylketonuria firstly analyzed by a Norwegian physician, Ivar Asbjorn Folling in 1934. He once analyzed two Egeland siblings’ urine that came up with mental retardation and found that there are compounds which contained a benzene ring in both of the urine (later known as phenylpurivic acid). After that he requested other physician near Oslo to do the same urine test for their mental retardated patients. The results have shown that among 430 children tested there are eight others had the same abnormality as the Egeland children, including two more other sibling pairs.3

After that, for years PKU was considered as an unfortunate mental retardation that could not be treated. But in the mid 1950’s, it was developed a low-phenylalanine diet formula that could prevent the mental retardation of PKU.3 Then in the 1960‘s Guthrie found the bacterial inhibitation assay for screening of PKU for newborn baby. Until now, researches are still remained to find the effective treatment of the PKU.

III. Incidence

The occurrence of PKU varies among population worldwide.1,2 Such as in the United States, PKU occurs in 1 in 10,000 to 15,000 newborns.1 A high incidence is reported in Turkey (approximately 1 in 2600 births), the Yemenite Jewish population (1/5300), Scotland (1:5300), Estonia (1:8090), Hungary (1/11,000), Denmark (1/12,000), France (1/13,500), the United Kingdom (1/14,300), Norway (1/14,500), China (1/17,000), Italy (1/17,000), Canada (1/20,000), Minas Gerais State in Brazil (1/20,000),and the former Yugoslavia (1/25,042). A low incidence is reported in Finland (< 1/100,000)and Japan (1/125,000).2

IV. Etiology

PKU is an autosomal recessive disorder caused by mutations in the PAH gene.1,2 This PAH gene is located on 12q23.2, spans about 171 kb and contains 13 exons. There are more than 500 different mutations in the PAH gene have been identified, and it can be of various types, including missense mutations (62% of PAH alleles), small or large deletions (13%), splicing defects (11%), silent polymorphisms (6%), nonsense mutations (5%), and insertions (2%).1 The PKU that is caused by this PAH gene mutation commonly called as classical PKU.1-3 The PAH gene provides instructions for making phenylalanine hydroxylase enzyme that converts the amino acid phenylalanine to be tyrosine.1-2 The gene mutations reduce the activity of phenylalanine hydroxylase, leads to build up the phenylalanine to toxic levels in the blood and other tissues.2 Because nerve cells in the brain are particularly sensitive to phenylalanine levels, excessive amounts of this substance can cause brain damage.1-3

PKU is an autosomal recessive genetic disorder, which means that both parents must have at least one mutated allele of the PAH gene inherited and cause the PKU to their child. The child must inherit both mutated alleles, one from each parent. So, it is still possible for a parent with the disease to have a normal child if his wife or her husband has normal functional allele of the gene. Yet, a child from two parents with PKU will inherit two mutated alleles any time, and therefore the disease.

Besides the gene that express the PAH enzyme, there are other causes of PKU include BH4 deficiency and DHPR deficiency. BH4 deficiency is caused by mutated alleles at 3 other loci (11q22.3-23.3, 10q22, and 2p13) and DHPR deficiency involves abnormalities localized to 4p15.1-16.1.1

The first PKU mutation identified in the PAH gene was a single base change (GT>AT) in the canonical 5-prime splice donor site of intron 12 (612349.0001). (DiLella et al.,1986). Eisensmith and Woo (1992) reviewed mutations and polymorphisms in the human PAH gene. About 50 of the mutations were single-base substitutions, including 6 nonsense mutations and 8 splicing mutations, with the remainder being missense mutations. Of the missense mutations, 12 apparently resulted from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutations had been observed at several sites, producing associations with different haplotypes in different populations. Studies of in vitro expression showed significant correlations between residual PAH activity and severity of the disease phenotype.

V.    Pathophysiology

In most patients, the classic type of PKU involves a deficiency of PAH that leads to increased levels of phenylalanine in the plasma (>1200 µmol/L; reference range, 35-90 µmol/L) and to excretion of phenylpyruvic acid (approximately 1 g/d) and phenylacetic acid in the urine.1 PAH catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine.1-3

In normal people, phenylalanine in the blood is always in low amounts (approximately between 1-2 mgs%). As soon as infants with PKU began to take milk (breastmilk or cowmilk), the amount begins to increase. Within weeks, it reaches 20 to 30 times higher than normal.3 Classic PKU, the most severe form of the disorder, occurs when phenylalanine hydroxylase activity is severely reduced or absent. People with untreated classic PKU have levels of phenylalanine high enough to cause severe brain damage and other serious medical problems.1

Since the converting of amino acid phenylalanine into tyrosine involves a cofactor called tetrahydrobiopterin (BH4), another form of hyperphenylalaninemia will occur if there is a defect in the biosynthesis or recycling of the cofactor. In that case, the PAH level is normal but not act properly. BH4 is also a cofactor in the production of L-DOPA from Tyrosine and 5-Hydroxy-L-Tryptophan from Tryptophan, which must also be supplemented as treatment in addition to the supplements for Classical PKU. Levels of dopamine can be used to distinguish between these two types. Tetrahydrobiopterin is required to convert phenylalanine to tyrosine, but it is also required to convert tyrosine to L-DOPA (via the enzyme tyrosine hydroxylase), which in turn is converted to dopamine. Low levels of dopamine lead to high levels of prolactin. By contrast, in classical PKU, prolactin levels would be relatively normal.

Phenylalanine is a large, neutral amino acid (LNAA). LNAAs compete for transport across the blood-brain barrier (BBB) via the large neutral amino acid transporter (LNAAT). If phenylalanine is in excess in the blood, it will saturate the transporter. Excessive levels of phenylalanine tend to decrease the levels of other LNAAs in the brain. However, as these amino acids are necessary for protein and neurotransmitter synthesis, Phe buildup hinders the development of the brain, causing mental retardation.

VI.       Clinical Manifestation

The clinical manifestations of PKU are caused by the excess of phenylalanine and deficient of tyrosine. Infants with PKU appear normal in birth until a few months old later. Without treatment with a special low-phenylalanine diet, these children develop permanent intellectual disability. Seizures, epilepsy, developmental delay, behavioral problems, and psychiatric disorders are also common. Untreated individuals may have a musty or mouse-like odor as a side effect of excess phenylalanine in the body. Children with classic PKU tend to have lighter skin and hair than unaffected family members and are also likely to have skin disorders such as eczema. Skin finding due to the impairment of melanin synthesis: hypopigmentation, light sensitivity, keratosis pilaris, pyogenic infection, etc.1,2

Babies born to mothers with PKU and uncontrolled phenylalanine levels (PKU women who no longer follow a low-phenylalanine diet) have a significant risk of intellectual disability because they are exposed to very high levels of phenylalanine before birth. These infants may also have a low birth weight and have slower growth than other children. Other manifestations include heart defects or other heart problems, an abnormally small circumference of head (microcephaly), and behavioral problems. They will be a destructive and irritable person.1,2

VII.             Screening and Additional Examination

Prenatal diagnosis and screening can be carried out by several methods.4 For over 25 years, we have used the Guthrie bacterial inhibition assay (GBIA) for this purpose and report our results in comparison with amino acid analyzer (AAA) results. Methods: Plasma phenylalanine was measured by the amino acid analyzer and blood phenylalanine in dried blood spots was measured by GBIA in paired specimens. Recording of values was performed blinded. Newborn screening to identify patients with metabolic disorders was pioneered by Robert Guthrie in the 1960s for the detection of PKU. Tandem mass spectrometry (MS/MS) has now been applied to newborn screening, expanding the ability to screen for 50 different metabolic diseases. 4

VIII.       Treatment

Since its discovery, there have been many advances in the treatment of PKU. It can now be successfully managed by the patient under ongoing medical supervision to avoid the more serious side effects. Overall, the management for PKU patients can be divided into 3:

  1. Diet

Early cases of PKU were treated with a low-phenylalanine diet. More recent research has now shown that diet alone may not be enough to prevent the negative effects of elevated phenylalanine levels. Optimal treatment involves maintaining blood Phe levels in a safe range while monitoring diet and cognitive development. If PKU is diagnosed early enough, an affected newborn can grow up with normal brain development, but only by managing and controlling Phe levels through diet, or a combination of diet and medication. Optimal health ranges are between 120 and 360 µmol/L, and aimed to be achieved during at least the first 10 years. Restriction should be observed before pregnancy, during pregnancy and during breast-feeding to avoid the harmful effect to nervous system.2, 3, 5

The diet requires severely restricting or eliminating foods high in Phe (for example meat, chicken, fish, eggs, nuts, cheese, legumes, milk). Starchy foods, such as potatoes, bread, pasta, and corn, must be monitored. Infants may still be breastfed to provide all of the benefits of breastmilk, but the quantity must also be monitored. The sweetener aspartame that present in many diet foods and soft drinks must also be avoided. As the child grows up these can be replaced with pills, formulas, and specially formulated foods. Tyrosine, which is normally derived from phenylalanine, must be supplemented.5

2. Enzyme replacement therapy

An alternative treatment for PKU is enzyme substitution therapy with a recombinant phenylalanine ammonia lyase (PAL). PAL is a bacteria-derived enzyme that catalyses the conversion of L-phenylalanine to transcinamic acid and ammonia without a cofactor requirement. Subcutaneous delivery of PAL to hyperphenylalaninemic mice models successfully converted Phe to harmless metabolites. However PALs metabolite effect is not sustained due to immune responses and Phe levels have to be stringently monitored to prevent hypophenylalanemia.5

3. Gene therapy

Gene therapy is an experimental, yet very promising approach for PKU treatment. By delivering a functional PAH gene to the liver in vivo, its activity should be reconstituted leading to normal clearance of Phe in the blood therefore eliminating the need for dietary restrictions or frequent enzyme replacement therapies. Significantly, it has been reported that reconstitution of 10-20% of normal PAH enzymatic activity is sufficient to restore normal serum Phe levels. The advanced researches in PKU treatment by gene therapy have been accelerated by the availability of pre-clinical models of disease.

The availability of a mutant mouse that closely mimics the human disease, called PAHenu2 provides an ideal model for investigating gene transfer in vivo and invaluable information on the pathology and biology of PKU. The original PAHenu2 mouse was generated by chemical mutagenesis of a BTBR-mouse strain using alklating agent ENU, resulting in a mutation in the PAH gene called PAHenu2. Numerous genetic and biochemistry studies have confirmed the reliability of this mouse model to closely resemble the metabolic and neurobiological phenotype of human PKU.


  1. OMIM. Phenylketonuria. [article on the internet]. 2010. Available from: URL:
  2. Steiner RD. Phenylketonuria. [article on the internet] 2011. Available from:
  3. Use of the Guthrie bacterial inhibition assay to monitor blood phenylalanine for dietary treatment of phenylketonuria. Screening, Vol 4, Issue 4. 1996. p.205-11.
  4. Redei GP. Encyclopedia of genetics, genomics, proteomics and informatics. 3rd edition. 2008. Columbia: Springer
  5. Argyros O, Wong SP. Gene therapy for phenylketonuria. [article on the internet]. Available from: URL:
  6. Hanley WB, Demshar H, Preston MA, Borczyk A, Schoonheyt WE, Clarke JT, et al. Newborn phenylketonuria (PKU) Guthrie (BIA) screening and early hospital discharge. Clinical Chemistry. 2008. 54:12.1961–8.
  7. Wappner R, Cho S, Kronmal RA, Schuett V, Seashore MR. Management of phenylketonuria for optimal outcome: A review of guideline for phenylketonuria management and a report of surveys of parents, patients, and clinic directors. Pediatrics 1999;104; e68 DOI: 10.1542/peds.104.6.e68.
  8. Choi JO, Park JW, Oh HJ, Seo KI, ParkHY, Jung SC. Gene therapy for phenylketonuria mouse model using pseudotyped adeno-associated virus vector. Gene Therapy. 2006. 13, 587–593. doi:10.1038/

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