Phenylketonuria: Issues and Research

Introduction

This paper will address the Genetic and Metabolic Disorder known as Phenylketonuria. After the introduction, this paper is divided into seven main sections. Each section will address a number of issues related to this illness.

PKU: Causes and Effects

Phenylketonuria (PKU) is an inherited disorder affecting the metabolization of amino acids.1 PKU is caused by mutations in the phenylalanine hydroxylase (PAH) gene which inhibits the body's ability to breakdown phenylalanine (Phe) into Tyrosine. Thus this hereditary disease is also monogenic. Phe is an essential amino acid that is found in all proteins in varying quantities.1 Tyrosine is Phe's metabolite and a precursor of dopamine and other crucial brain neurotransmitters.1(59) When large quantities of Phe accrete in the body it has a neurotoxic effect. This effect causes the brain to undergo structural damage. This, in turn, can lead to both severe mental retardation and psychiatric disturbances if left untreated.

Indeed, research has been conducted demonstrating the connection between PKU and such psychiatric diseases as depression, generalized anxiety disorder, social phobia, social maturity problems, and social isolation issues.1(59) Brumm, Bilder, and Weisbren report that PKU has also led to severe developmental disorders in children. These problems include attention deficit, a lack of social competence, a lack of self-sufficiency, and poor self-esteem.1(59) Those who suffer from PKU usually experience lifelong struggles with one or more of these problems.

As noted above, PKU is a disease that affects the normal cognitive and physical development of children. It was first discovered as a cause in human development in 1953.2 Since 1963 all pregnant mothers have undergone neonatal screening to detect the disease. After birth, the at-risk child is placed on a restricted diet to reduce the disease's effects.3

Treatment and Related Issues

The treatment for PKU involves reducing the amount of protein in the diet as much as possible. This has to be accomplished without affecting the normal progression of development. One possible way to consistently evaluate the disease progression is through the use of metabolic network measurements - a protocol that has been used in evaluating the progression of Huntingdon's disease. In turn, to replace the reduced protein, a diet high in carbohydrates is substituted.4(662) A key difficulty with the treatment regimen is the social burden it creates.2(S91) It's reportedly quite difficult for a patient to maintain this diet and so recent research has focused on alternative solutions. There will be more discussion of this in the section on future research. At the present time, no other lifestyle factors, other than diet, have been identified as efficacious.

It is pertinent to note that, long-term deviation from the diet creates significant health risks for all patients affected by the illness. This includes adults, who may find a life-time diet requiring low consumption of protein difficult to maintain. Unfortunately, the health consequences of treatment deviation are as serious for adults as for children and include irreversible brain damage. This also creates a special burden on parents with infants and children suffering from PKU. It requires special diligence to keep the effects of the disease at bay. As there is no cure for this disease, the dietary treatment regime will need to be observed over the entire lifetime of the patient.2(S91), 3(689), 5(109)

An important component of this lifetime treatment is the importance of monitoring. Monitoring involves testing for levels of Phe in the blood. However, until relatively recently monitoring was only possible by means of laboratory tests which involved a waiting period for results. This waiting period allows crucial time to elapse before necessary assessments and interventions can take place.

This has been mitigated with the introduction of portable monitoring devices. Home testing now allows for the patient to receive results in real-time. 2(S91) As always, patients are required to test their blood Phe levels on a regular basis. For children, tests from two to three times per week are required. The frequency of testing declines as the patient ages and can fall to once per week for older teenagers. For adults, testing is usually recommended at once per month.6

For children that successfully undertake the treatment program, research reports that developmental difficulties are mostly abated. However, even in such patients, important developmental differences between PKU and non-PKU segments of the population have still been observed. For instance, van Spronsen and Enns2(S91) reports a 5-7 point difference in IQ scores between the two populations. As noted above, PKU sufferers also experience serious psychiatric problems as well.

Therefore, physicians who are treating PKU must also take its psychiatric and psychological effects into consideration. It's likely that PKU treatment can lead to a significant abatement of some, but not all, symptoms. Thus it's also necessary to treat any co-morbid psychiatric conditions that arise. In the latter case, treatments may likely recommend the use of psychiatric mediations. At this time, there is not much research examining potential drug interaction effects between the use of such drugs and PKU treatment.1(S60) This is an area for future recommended research. It's enough to say, that interaction issues are not well understood at the present time. However, psychiatric drugs do affect the same areas of the brain as PKU affects. This includes regions of the brain that regulate mood, emotion, and cognition.1(S60)

There has also been some research that links PKU treatment with an increase in obesity. However, this result has not been confirmed by all research. In fact, Rocha et al report that PKU patients actually have a lower incidence of obesity than the general population. This is likely due to the low protein diet which is high in vegetables. 4(662)

Incidence

Despite the use of newborn screening since 1963, information regarding PKU incidence rates is reportedly sparse.6 The Council of Regional Networks for Genetic Services (CORN) supplies the data used to assess incidence rates. This data is based on the Newborn Screening Report published in 1994. The prevalence of PKU and non-PKU hyperphenylalaninemia can only be estimated using this data.6 According to this data, PKU prevalence falls into the range of 1 per 13,500 to 1 per 19,000 newborns.6 It's been reported that there are diverse differences in PKU state reporting to the NIH. The NIH thus reports a blended national estimate for PKU incidence of 1 per 48,000. There are also notable ethnic and racial differences in PKU distribution. The report found White and Native Americans had the highest incidence rates. The groups with the lowest prevalence are Hispanic, Asian and Black Americans.

Another difficulty, concerning PKU incidence and diagnosis, is that standards vary among different reporting authorities. In the US, there is some disagreement on what blood Phe level should be used to diagnose the disease. This has also been reported internationally where disagreements among many countries exist on what blood Phe level should trigger treatment.6, 5(109)

Hereditary, Genetic, and Environmental Factors

As noted above, PKU is a monogenic hereditary illness. Patients who suffer from the disease are experiencing a severe impairment of normal enzyme function. The NIH 6 defines PKU as a disease where an individual has elevated blood Phe levels on the order of > 20mg/dl and has accumulated phenylketones. An individual with partial PAH deficiency will experience a condition called non-PKU hyperphenylalaninemia. This involves a lower Phe blood level and an absence of accumulated phenylketones. In either case, the hyperphenylalaninemia condition is an autosomal recessive disorder. Its cause is due to mutations in the PAH gene.6 It is rare that mutations in other different genes result in any form of hyperphenylalaninemia.6 However, there are cases where genes needed to synthesize or recycle the tetrahydrobiopterin cofactor of PAH result in a similar pathology.6 It should be noted, the implicated chromosome has been identified as 12q22-24.1.3(387)

Although PKU is monogenic and not multifactorial, it does exhibit considerable genetic variability. The PAH gene has been associated with as many as 400 different mutations.6 These mutations take the form of deletions, insertions, missense mutations, splicing defects, and nonsense mutations. Most PKU sufferers are compound heterozygotes. This creates the possibility for multiple genotypic combinations. It also contributes to the clinical heterogeneity of the illness. The mutations are also implicated in the disease's biochemical heterogeneity. It's also possible mutations are the cause of the biochemical phenotype.

The phenotype's genetic contributions are quite complex. These contributions consist of documented allelic heterogeneity in the PAH gene.6 Some PAH alleles are associated with PKU and some with the variant non-PKU hyperphenylalaninemia. Genes at other loci may impact the transport of Phe within the brain. These other genes may also affect the size and metabolic control of the Phe pool.6 PKU’s molecular heterogeneity causes wide phenotypic heterogeneity and contributes to biochemical uniqueness. Enzymatic activity may be predictable based on PAH phenotype. However, the clinical phenotype and the genotype relationship sometimes varies.

Thus, the fact that discordant phenotypes among siblings who possess the same genotype at the PAH locus exist is suggestive. It implicates other genetic and environmental factors in clinical phenotype influence. While this suggests the existence of important modifier genes, these genes have reportedly not been identified as yet.6 It has been reported that a small cohort of PKU patients, who are not on treatment, have not exhibited any mental retardation. This suggests some variance in Phe transport into the brain and thus variance in clinical symptoms and degree of pathology.6

In terms of environmental and lifestyle factors, variance between individuals with genetically identical mutations has been explained by a number of factors. These factors include age at diagnosis, age at commencement of metabolic control, and degree of metabolic control.6 Observed variations are dependent on the individual trait under examination. Examples of such traits include mental retardation in untreated cases, blood Phe level, neurological and neuropsychiatric deficits, or brain Phe concentration.6 Due to the fact that PKU treatment was begun in 1963, there is no reported data yet on the clinical manifestations of PKU as early treated individuals age. That is, not many PKU treated patients are yet over the age of 40.6 This suggests that new clinical characteristics of PKU may still be discovered. At the present time, it's not possible to predict future clinical results of PKU treatment.

It's worth mentioning that many common health disorders are caused by multiple genes, an interaction between genes and environmental factors, or interactions between multiple genes. Although PKU is monogenic, it's still a complex disease because of its variability.9

PKU Diagnostic Testing

As noted above, neonatal screening is the method used to diagnose PKU in newborns. Thus it's crucial for a patient to be seen by a medical professional in order to obtain a diagnosis. The screening process involves collecting neonatal blood samples on special paper cards. The collection is taken during the newborn's first days after birth. The samples are then tested for the presence of whatever blood levels of Phe testing authorities deem abnormal. Infants who are found to have abnormally high levels of Phe are immediately referred for comprehensive treatment.6

There are three principal techniques used for PKU screening of newborns in the US. These techniques are all population-based. The first technique is the Guthrie Bacterial Inhibition Assay (BIA). The second technique is fluorometric analysis. The third technique is tandem mass spectrometry.6 Of these, the fluorometric analysis and tandem mass spectrometry are measurable and programmable. When compared to BIA, these methods are likely to result in a lower number of false positives. The tandem mass spectrometry can obtain data relevant to the elevation of tyrosine that's useful in Phe levels analysis.6

It should be noted that diagnosis is not always straightforward. This is because the concentration of phenylalanine in the blood may not have reached its maximum level. It's also suggestive that a single high test may be anomalous. Therefore, some researchers recommend multiple testing over a period of weeks to inform the diagnosis, rather than rely on a single test.8

Ethical, social and legal issues

The main ethical issues that may be at play in PKU treatment concern the treatment of children dependent upon the due diligence of a caregiver. If a parent lapses in dietary treatment it has been shown to have severe consequences. It may be that parents who are not particularly good caregivers may neglect their child's treatment. How our society's institutions address such an issue is an area of ethical concern. It has been noted above that PKU creates a serious social burden. It's unclear from the research what legal issues are involved in PKU diagnosis or treatment. Although the wide variance in diagnostic standards among testing authorities could lead to lawsuits if an opportunity to treat is missed. It's not known from this literature review whether this has ever occurred either in the US or internationally.

Nutrition, Supplemental Formulas and Resources

As noted above, infants with PKU require additional attention as to the content of their daily diet. The focus of PKU treatment involves such elements as the consumption of concentrated protein substitutes, measurement of phenylalanine exchanges, and promoting low phenylalanine nutrition sources.11 Some weaning guidelines are not compatible with the nutrition needs of an infant afflicted with PKU.11 Thus health professionals approximate the nutrition guidelines of non-PKU infants as much as possible.11 It also bears repeating that a single international standard of weaning guidelines for infants with PKU doesn't exist at the present time.

Infants newly diagnosed with PKU receive an age-specific phenylalanine-free protein substitute as well as either breast milk or standard formula. During weaning's early phases, PKU affected infants take in most of the nutrition and protein in their diet from phenylalanine-free infant formula, along with a protein substitute, breast milk or standard formula. Then small amounts of solid food are gradually introduced into the infant's diet.11 These low protein items include fruit purees and vegetables having low amounts of protein (such as carrot and sweet potato). Also included in the diet are low protein cereal and commercial baby foods that contain less than 0.5g of protein per 100g. When the foods are consistently accepted by the infant, the solids are expanded further. These foods are measured 50mg phenylalanine exchanges that include 1g of natural protein.11 This adjustment in the diet involves progressive reductions in phenylalanine sourced from breast milk or standard formula. By age six to 12 months, phenylalanine free infant protein substitute is not considered adequate enough to fulfill the non-phenylalanine protein dietary requirement. As such, supplementation using the more concentrated form of phenylalanine-free protein substitute is necessary.

For young children, the phenylalanine-free protein substitutes are in powdered form and intended to be mixed with liquid before ingesting. However, the design of such powders somewhat neglects the nutritional needs or feeding development of infants older than six months.11 The administration of large volume liquid-form protein substitutes can have the effect of delaying the progression to solid food consumption. This is because these powders have a suppressing effect on appetite.11 The challenges administering protein substitutes have been reported and that compliance is below required nutritional levels.

Indeed, a UK study on children living with PKU, between the ages of 1 and 5, found the administering of liquid protein substitutes was connected with many negative behaviors. These behaviors included crying, screaming, gagging, and spilling containers containing the protein substitute drink. The mental state of caregivers assessed during these events was reported as stressful.11 A study, on children with PKU aged from six months to ten years, suggested an alternative approach replacing the liquid substitute with a paste.11 The paste was found to be much more palatable to the children. Another difficulty with administering protein substitutes is the bitter taste.11 However, some research found early and repeated exposure to bitter-tasting foods raised the likelihood of acceptance by the child. 11 This likelihood increased if the caregiver was also observed ingesting the bitter-tasting food. Protein substitutes should be given in equal portions and at regular intervals throughout the day.

Nutrition resources, for use by health professionals and caregivers of children living with PKU, include national guidelines and dietary plans for the administration of food. Table 1 below includes an example of such a dietary plan in use in the UK.11 It includes recommendations for achieving protein and energy requirements during the weaning period for infants living with PKU.

(Table 1 omitted for preview. Available via download)

(Figure 1 omitted for preview. Available via download)

Alternative and Emerging Medical Treatments

As noted above, the standard medical treatment for a newly diagnosed patient is a diet with low levels of protein. It's widely acknowledged, that while efficacious for treatment, protein is an essential ingredient in the diet of both children and adults. Therefore, research has focused on areas in which the societal burden can be reduced for the patient. Another area is how protein can be included in the patient's diet without causing harm. It bears reiterating that while diet is the standard treatment, it doesn't produce completely satisfactory results. This section will consider some of these new treatments. These treatments are focused on addressing nutritional awareness issues from a number of different perspectives. These perspectives include at the level of dietary intake, at the gut level, in the liver, in the genes and cells of the body, and in the brain.2(S91-S93) It should be noted that the amount of research on future treatments is considerable in scope. Due to space limitations only some of the ongoing or completed research methodologies are included.

Dietary intake. As noted, issues with protein substitutes are key in a PKU influenced diet. However, recent research has found evidence of a protein with low enough levels of Phe to possibly be palatable to a patient undergoing treatment. The protein has been identified as glycomacropeptide (GMP) and it's a byproduct from goat's milk acquired during the cheese-making process. One drawback of protein substitutes are complaints as to their palatability. GMP would seem to be an improvement over these substitutes. However, GMP is not completely devoid of Phe, therefore, it has only limited utility. GMP is also devoid of other amino acids, such as tyrosine, which must be added.2(S91-S93)

Gut level. The gut may be useful in PKU treatment in two ways. First, the phenylalanine-ammonia lyase (PAL) enzyme is active in the gut if taken orally.2(S91-S93) Although it must be ingested in a non-absorbable and protected form.2(S91) The enzyme metabolizes Phe into a non-toxic compound called trans-cinnamic acid with ammonia as another byproduct. It should be noted, the amount of ammonia is small and is easily converted by the body into urea. The enzyme's activity can be increased if it's injected into the patient in a form that's PEGylated. Note, PEGylation occurs through the process of attaching polyethylene glycol polymer chains to PAL.2(S91) Preclinical trials using the injectable PEGylated form of PAL are reportedly promising. Results from the trials show that Phe levels were reduced in both brain matter and in the vascular space. Also, PKU symptoms were reduced noticeably. Another promising aspect of this treatment is that PEGylation permits for subcutaneous depot injection of PEG-PAL. This is a type of enzyme delivery system that can produce relatively long-acting effects.2(S92) Clinical trials were reportedly begun in 2008, however, there is no additional information to report about the study at this time.

The second way PKU treatment can work at the gut level is by supplementation using large neutral amino acids (LNAA). LNAA are known to produce primary and secondary effects related to Phe concentrations. The first effect reduces Phe concentration in brain matter. The secondary effect involves reduction of Phe concentration in the blood.2(S92) The treatment is based on the discovery that Phe and other LNAA share the same transport system. This creates a competitive inhibition of the transport of LNAA with each other. Thus, supplementation would work by counteracting the effects of high-level Phe concentrations in the body's transport system.

Liver. The liver is important in Phe treatment because the main related metabolic activity takes place at this site. It was discovered in 1999 when a patient with end-stage liver disease underwent a liver transplant. The transplant had the effect of completely correcting the metabolic phenotype.2(S92) In addition, some patients who underwent successful dietary treatment still show evidence of cognitive impairment. It was noted that this minority of PKU patients responded poorly to the standard treatment even though it adequately controlled Phe in their blood. It was found these patients shared a common deficiency of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). This deficiency was believed to be caused by an impairment in the body's ability to synthesize or recycle BH4. A loading test was used to differentiate those PKU patients with this deficiency. Following this initial demonstration 6R- BH4., more formerly known as sapropterin dihydrochloride, and copyrighted under Kuvan, was developed from a chemical compound into a drug that received FDA and EMEA approval. It's been administered since 2008 in patients with mild PKU having the crucial BH4.2(S92),7

Genes and cells. Experimental protocols to treat PKU using gene therapy were developed following research that demonstrated that only 10 percent enzyme activity was necessary for Phe metabolism in mice studies. However, some examples of using gene therapy in humans have experienced serious complications. The efficacy of using gene therapy to treat PKU appears to still be under investigation.2(S92)

At the cellular level, hepatocyte transplantation is currently being studied. In this case, donor cells are efficacious when they enjoy a certain benefit relative to host hepatocytes. This benefit is of a selective growth nature. This method has been researched in preclinical studies using both human and animal models. However, this technique has yet to be used to treat PKU. It is a potential avenue for future research still under consideration.2(S92) Vineis and Pearce10 report that PKU transmission could be avoided during cell communication by blocking the transmission of the PKU mutation to one's descendants. It's unclear if there is any viable research testing this as a potential treatment.

Brain level. Although the effects of PKU on brain activity are currently well understood, its pathogenesis in brain damage is not. It has been observed that the only effects of PKU are in the brain. The brain, in turn, is separated from the circulatory system, by the brain-blood barrier (BBB). This lends credence to the notion that the BBB is of critical importance in understanding PKU pathogenesis.2(S92) The essential workaround using the brain involves treatments that can cross the BBB. These studies involve the use of BH4, which can cross the BBB. But at the present time, the research in this area is still inconclusive as to whether treatment regimens using BH4 would successfully cross the BBB.

References

1. Brumm, V.L., Bilder, D., Waisbren, S.E. Psychiatric symptoms and disorders in phenylketonuria. Mol Genet Metab, 2010; 99: S59–S63.

2. Van Spronsen, F.J., Enns, G.M. Future treatment strategies in Phenylketonuria. Mol Genet Metab, 2010; 99:S90-S95.

3. Lamônica, D.A.C., Stump, M.V., Pedro, K.P., Rolim-Liporacci, M.C., Caldeira, A.C.G.C., Anastácio-Pessan, F., et al. Breastfeeding follow-up in the treatment of children with Phenylketonuria. J Soc Bras Fonoaudiol, 2012; 24:3816-9.

4. Rocha, J.C., van Spronsen, F.C., Almeida, M.F., Soares, G., Quelhas, D., Ramos, E., et al. Dietary treatment in phenylketonuria does not lead to increased risk of obesity or metabolic syndrome. Mol Genet Metab, 2012; 107: 659-663.

5. Blau, N., Belanger-Quintana, A., Demirkol, M., Feillet, F., Giovanni, M., MacDonald, A., et al. Management of Phenylketonuria in Europe: Survey results from 19 countries. Mol Genet Metab, 2010; 99:109-115.

6. Phenylketonuria: Screening and Management. NIH Consensus Statement 2000 October 16-18; 17(3): 1-27. Retrieved from http://consensus.nih.gov/2000/2000phenylketonuria113html.htm. Oct. 2013.

7. Lindegren, M.L., Krishnaswami, S., Reimschisel, T., Fonnesbeck, C., Sathe, N.A., McPheeters, M.L. A systematic review of BH4 (Sapropterin) for the adjuvant treatment of Phenylketonuria. JIMD Rep., 2013; 8:109–119.

8. Blau, N., van Spronsen, F.J., Levy, H.L. Authors’ reply. The Lancet, 2011; 377:466.

9. Becker, F., van El, C.G., Ibarreta, D., Zika, E., Hogarth, S., Borry, P., et al. Genetic testing and common disorders in a public health framework: How to assess relevance and possibilities. Eur J Med Genet, 2011; 19: S6–S44.

10. Vineis, P., Pearce, N.E. Genome-wide association studies may be misinterpreted: genes versus heritability. Carcinogenesis, 2011; 32:1295-1298.

11. MacDonald, A., Evans, S., Cochrane, B., Wildgoose, J. Weaning infants with phenylketonuria: a review. Journal of Human Nutrition and Dietetics, 2012; 25(2): 103-110.