By Nariman Hossein-Javaheri

Phenylketonuria,also known as PKU, is an autosomal recessive disorder in which a child is born with inability to metabolize
a specific amino acid, phenylalanine. This digestion incapability is due to a mutation in PAH gene from which the enzyme phenylalanine hydroxylase (PAH) is created. The active form of this enzyme converts phenylalanine to tyrosine and many other components important for the body. If PAH is not functional, phenylalanine cannot be digested which will lead to an increase in the concentration of this substance in blood. Excess amounts of phenylalanine are then converted to phenylpyruvate and the resulting high accumulations of phenylpyruvate can be very toxic(6). This toxicity affects neuronal cells the greatest especially during early stages of childhood and development causing intellectual disabilities, mental retardations and other serious health problems. In addition to mental impairment, PKU has several visible symptoms including seizures, tremors and jerky movements in arms or legs. Furthermore, affected patients have much lighter skin and hair color than their unaffected family members. It is important to know that phenylketonuria is a treatable disease in which the treatment involves a very low containing phenylalanine diet which must be strictly followed for the rest of one’s life. In this diet usage of any high phenylalanine containing substance such as meat, eggs and milk are greatly reduced(16).

Phenylketonuria detection test

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Figure1. Blood taken by Heel Prick in Guthrie test used to screen newborns with PKU.

Occurrence of PKU is very diverse among different human population where Middle Eastern countries such as Turkey and Iraq have the highest incidents of 1 in every 2500 child(1). In Canada approximately 250 new born babies are diagnosed with phenylketonuria annually as suggested by Canadian Mental Health Association therefore, it is important to detect this disease early after birth.
The most common test performed worldwide to detect PKU is the Guthrie Screening test also known as bacterial inhibition assay method, developed in 1966. The purpose of this test is to detect elevated levels of phenylalanine in infant’s blood. Guthrie Screening test utilizes phenylalanine’s property to facilitate bacterial growth in cellular cultures in contact with antibiotics. At first, a drop of blood is obtained by pricking infant’s heel and is collected using a filtered paper which is then, sent to laboratory for further examination. In the laboratory, the blood sample is placed on an agar gel containing bacteria and antibiotics. Concentration of both bacteria and antibiotics together are in equilibrium and bacterial growth and death are relatively stable. However, if the introduced blood sample has high concentration of phenylalanine, a shift in equilibrium will be observed with an increase in bacterial growth rate. This growth is visible with naked eye within a day. With such an increase in growth rate, infant is diagnosed with phenylketonuria(2,3).

Symptoms of phenylketonuria


PKU has several psychological and psychiatric symptoms associated with it. Such symptoms include attention problems, school problems, asociality and low self esteem with great decrease in motivation. Furthermore, such psychiatric problems can become more severe by evolving to depression, general anxiety disorder, elevated mood swings and bipolar disorders(4). It is important to know however, PKU does not only affect mental health but motor activity and movements are also greatly influenced. Such physical symptoms include smaller head size in comparison to normal, hyperactivity, jerky limb movements, seizures, tremors, skin rashes and unusual positioning of hands. Furthermore, affected patients have much lighter skin and hair color than their unaffected family members(5).

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Figure2. Patients with phenylketonuria

Pathophysiology of phenylketonuria

As mentioned previously, in Phenylketonuria, a child is born with inability to metabolize a specific amino acid, phenylalanine. This digestion incapability is due to a mutation in PAH gene from which the enzyme phenylalanine hydroxylase (PAH) is created. Active form of this enzyme converts phenylalanine to tyrosine and many other components important for the body. If PAH is not functional, phenylalanine cannot be digested which will lead to an increase in the concentration of this substance in blood. Excess amounts of phenylalanine are then converted to phenylpyruvate where high accumulations of phenylpyruvate can be very toxic. This is known as the classic phenylketonuria. Further studies however have indicated that another form or PKU exists in which not PAH but its coenzymes are inactive. The main inactive coenzyme causing PKU is Tetrahydrobiopterin (BH4)(6). Tetrahydrobiopterin is an essential cofactor for phenylalanine, tyrosine and tryptophan mono-oxygenases. Not much is known about cellular regulations of BH4 but several studies have shown that some patients suffering from PKU have decreased levels of tetrahydrobiopterin in their blood serum.Further studies have revealed that elevated levels of phenylalanine in blood will lead to an increase in the activity of BH4. Therefore, any mutation or change in structures of tetrahydrobiopterin can lead to a rare form of phenylketonuria. In order to distinguish between the classic and tetrahydrobiopterin induced phenylketonuria, concentration levels of the hormone prolactin are measured in the blood stream. Prolactin concentration greatly depends on dopamine (DA) concentrations regulated by BH4. It is important to know BH4 not only causes the transformation of phenylalanine to tyrosine in an indirect way but, it is also required to convert tyrosine to L-DOPAwhich is the precursor for DA. Hence, low activity of BH4 will lead to decreased L-DOPA concentrations and resulting in an eventual reduction in DA levels. Low DA concentrations will increase the concentration of the hormone prolactin. In classic PKU however, levels of prolactin will remain normal(7,8).


Genetic mutation in phenylketonuria


The PAH gene located on chromosome 12 (12q23.2), spans 171kb and contains 13 exons which encodes for the enzyme phenylalanine hydroxylase with 452 amino acids. Phenylketonuria arises from a mutation in PAH and follows and autosomal – recessive pattern of inheritance. Basic structure of human PAH gene is presented in figure 3.
Figure3. The basic structure of the human PAH gene. Found on the long arm of chromosome 12 (12q23.2), the human PAH gene contains 13 exons which encode a polypeptide of 452 amino acids.
Figure3. The basic structure of the human PAH gene. Found on the long arm of chromosome 12 (12q23.2), the human PAH gene contains 13 exons which encode a polypeptide of 452 amino acids.
mutations in PAH gene mostly result from an arginine111 to tyrosine111 in exon3 point mutation of the PAH gene.The mutation is in linkage disequilibrium with the mutant haplotype4 alleles which are the most prevalent haplotype among human population. Furthermore, it is important to know further mutations in different haplotypes can lead to PKU: A point mutation in exons 7, 9 and 12 can change amino acids from glutamate280 to lysine 280 (by changing guanine to adenine), leucine311 to proline311 (by changing thymine to cytosine) and arginine408 to tyrosine 408(by changing cytosine to thymine)
respectively (9).


Metabolic pathway affected in phenylketonuria


As mentioned previously, in PKU one is incapable of metabolizing phenylalanine which can lead to great toxicity for neuronal cells. There are two main pathways in which phenylalanine are generally metabolized. First and the most common path is phenylalanine (phe) oxidation to tyrosine. The second or the minor pathway is phe transamination to phenylpyruvate and subsequent further metabolism to phenylacetylglutamate, o-hydroxyphenylacetate and phenyl lactate .
Figure4. Phenylalanine metabolism pathways.
Figure4. Phenylalanine metabolism pathways.

The major metabolism pathway requires activity of phenylalanine hydroxylase (PAH). In phenylketonuria however, the PAH is deficient therefore, the minor path will be used for metabolism (10).Using of the minor instead of the major pathway for phenylalanine metabolism will lead to a rise in levels of phenylacetylglutamate where it’s been know that this substance is very toxic and on its own, can lead to alteration in mental status and cognitive impairments if it is accumulated(11). In addition, studies have shown that other substances created in the minor pathway including phenyl lactate , can also lead to growth retardations by causing deficit of myelin in the cerebral hemispheres and the cerebellum and therefore, it has been directly linked to the mental retardations in PKU (12).Please refer to figure4.


Neuronal structural changes associated with phenylketonuria


Phenylketonuria can lead to severe psychological impairment and physical dysfunctions. These problems occur because of damage to the neuronal cells in the central nervous system. It has been suggested that both white and gray matters are affected in PKU.

White matter abnormalities


PKU can greatly affect myelination and white matter tracts in early stages of development therefore, it has been suggested that myelination or de-myelination can be the primary cause of neurological disorders associated with phenylketonuria. Both human and animal studies have shown a decrease in myelination levels of extrapyramidal neurons and glial cells where this reduction is caused by the transformation of oligodendrocytes to non-myelinating phenotypes. This loss of white matter is more visible in periventricular regions and the forceps major and minor of the corpus callosum in cortex(13). In addition not only myelination but also dendrite growth is impaired in PKU during early stages of development. However not much information is available on cortical changes related to white matter in humans since detecting dendritic growth and myelination are not achieved easily(14).

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Figure5. Statistical parametric t map showing relative decreases of white matter volume in patients with phenylketonuria.R indicates the right hemisphere, and the color bar represents the t score. Great decrease in periventricular white matter of the right hemisphere, involving the anterior forceps of the corpus callosum is observed.



Grey matter abnormalities

Abnormalities in grey matter in phenylketonuria can be detected more easily in comparison with white matter. Recent studies have shown a reduction in gray matter volume in patients suffering from PKU. This loss is most prominent in motor and pre-motor cortex, thalamus and the hippocampus. Interestingly, in certain studies, an increase in gray matter volume is observed in ventral regions of striatum.Gray matter abnormalities in such regions can explain for the occurrence of PKU symptoms where impaired motor and pre-motor cortex can cause impaired motor functions and volumetric reduction of neuronal cells in the thalamus and hippocampus and explain for mental and cognitive abnormalities(15).



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Figure6. Statistical parametric t map of gray matter volume reduction in patients with phenylketonuria superimposed on normalized structural images in selected planes.R indicates the right hemisphere, and the color bar represents the t score. loss of grey matter is present bilaterally in the motor cortex, the left premotor cortex, and the left thalamus, extending to the adjacent midbrain.


Treatments for phenylketonuria


Phenylketonuria cannot be fully treated however using a low phenylalanine diet can normalize phenylalanine concentrations and can prevent neurological disorders to some extent. It is recommended that treatment for PKU must begin during early stage of development to achieve maximum neural growth and function during adulthood. The low diet phenylalanine suggests protein rich foods such as eggs, milk, cheese fish and any type of meat should be avoided. Furthermore, carbohydrate rich diets such as potatoes, bread, pasta and corn must be consumed carefully. Patients must remain under strict monitoring for the rest of their lives(10). In order to receive necessary amino acids but avoid phenylalanine, a specific formula, Lofenalac, is consumed containing very low concentrations of phenylalanine but rich in other amino acids, providing a good protein source for PKU patients. Newer methods of treatments used in phenylketonuria include administration of tetrahydrobiopterin (BH4) orally which can reduce high levels of phenylalanine in certain patients(17,18). Even though plenty of recent improvements have been made to treat this disease, further studies are required to maybe sufficiently cure phenylketonuria.

References




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