Disorders of phenylalanine and tyrosine metabolism

Alsharhan, Hind; Ficicioglu, Can

doi:10.3233/TRD-200049

Disorders of phenylalanine and tyrosine metabolism

Article type: Review Article

Authors: Alsharhan, Hind^{a; b} | Ficicioglu, Can^{a; c; *}

Affiliations: [a] The Children’s Hospital of Philadelphia, Division of Human Genetics and Metabolism, Philadelphia, PA, USA | [b] Assistant Professor of Pediatrics, Biochemical Genetics, Faculty of Medicine, Kuwait University, Kuwait | [c] Professor of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

Correspondence: [*] Correspondence to: Can Ficicioglu, Children’s Hospital of Philadelphia 3401 Civic Center Blvd. Philadelphia, PA 19104, USA. FICICIOGLU@email.chop.edu

Keywords: Phenylketonuria, tetrahydrobiopterin, tyrosinemia, NTBC, alkaptonuria, newborn screening, genetic testing

DOI: 10.3233/TRD-200049

Journal: Translational Science of Rare Diseases, vol. 5, no. 1-2, pp. 3-58, 2020

Published: 03 August 2020

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Abstract

This article provides a review of the inborn errors of phenylalanine and tyrosine metabolism including the diagnostic approach, dietary and pharmalogical management and emerging therapies. Hyperphenylalaninaemia results mainly from defects in either phenylalanine hydroxylase (PAH) (resulting in phenylketonuria (PKU)) or the production or recycling of tetrahydrobiopterin (BH₄). Untreated PKU results in irreversible neurocognitive impairment. Five inherited disorders of tyrosine metabolism are known, which include tyrosinemia type I, type II, type III, hawkinsinuria and alkaptonuria. Newborn screening for these disorders has enabled their early detection and decreased the associated morbidity and mortality.

PHENYLKETONURIA

Phenylketonuria (PKU) is an autosomal recessive disorder resulting from a deficiency of phenylalanine hydroxylase (PAH), which converts phenylalanine (Phe), an essential amino acid, to tyrosine (Fig. 1) [1]. PAH is one of the three aromatic amino acid hydroxylases that utilizes tetrahydrobiopterin (BH₄) as a cofactor. PKU occurs in all ethnic groups, although it is more common in individuals of Northern European and Native American ancestry. The incidence is lower in those of African American, Hispanic and Asian ancestry. The average incidence is about 1 in 10,000 in the United States and Europe, and it is even greater in other populations (∼1 : 2600 in Turkey and ∼1 : 4500 in Ireland) [2, 3].

Fig.1

Metabolic Pathway of Phenylalanine.

PKU is due to total or near total deficiency of PAH activity. Normally, PAH activity is detectable in the liver as early as the eighth week of fetal life, and it reaches adult levels by the second trimester [4–7]. The inability of the body to break down phenylalanine results in its accumulation as well as the accumulation of alternate pathway products including phenylacetate, phenyllactate, and phenylpyruvate.

PKU Classification

The clinical classification between its three subtypes is arbitrary and based on Phe levels on a normal diet and/or phenylalanine tolerance in diet (Table 1). Classical PKU: It is the most severe type, and phenylalanine levels are more than 1200 μmol/L (mean normal: 60 μmol/L) on a normal diet. These patients can tolerate less than 250–300 mg of dietary Phe per day. Moderate/Mild PKU: Phenylalanine levels are above 600 μmol/L but less than 1200 μmol/L on a normal diet. These patients can tolerate 350–600 mg of dietary Phe per day. Mild hyperphenylalaninemia: Phenylalanine levels ranges from 360–600 μmol/L on a normal diet.

Table 1

Inherited disorder of phenylalanine metabolism

Enzyme defects	Tissue distribution	Chromosomal localization	Disorder	Characteristic	Treatment
PAH deficiency	Liver, kidney	12q22–q24.1	*Classical PKU*	Plasma phenylalanine > 1200μmol/L	Phenylalanine restricted diet
*Moderate/Mild PKU*	Plasma phenylalanine > 600 and < 1200μmol/L	Phenylalanine restricted diet
*Mild hyperphenylalaninemia*	Plasma phenylalanine < 600μmol/L	Regular diet*
GTP cyclohydrolase I (GTPCH) deficiency	Liver, brain, kidney, lymphocytes	14q22.1–q22.2	Tetrahydrobiopterin Deficiency	dopa-responsive dystonia	Levodopa/DCI
Autosomal dominant form
Tetrahydrobiopterin Deficiency	Seizures, hypotonia, tremor, developmental delay, hyperphenylalaninemia, very low neopterin and biopterin	Low-phe diet, BH4, L-dopa/carbidopa,5-OH-tyrptophan, folinic acid
Autosomal recessive form
6-Pyruvoyl-tetrahydropterin synthase deficiency	Liver, brain, kidney, lymphocytes, erythrocytes, fibroblasts	11q22.3–q23.3	Tetrahydrobiopterin Deficiency	Mental retardation, microcephaly, seizures, irritability, abnormal movements, hyperthermia, hyperphenylalaninemia, high neopterin, trace biopterin	Low-phe diet, BH4, L-dopa/carbidopa,5-OH-tyrptophan, folinic acid
Sepiapterin reductase (SR) deficiency	All tissues	2p14–p22	Tetrahydrobiopterin Deficiency	dopa-responsive dystonia, sepiapterin is present in urine, CSF	L-dopa/carbidopa,5-OH-tyrptophan, folinic acid
Dihydropteridine reductase (DHPR) deficiency	All tissues	4p15.3	Tetrahydrobiopterin Deficiency	Microcephaly, hypotonia, mental retardation, seizures, hyperphenylalaninemia, normal/high neopterin, very high biopterin	Low-phe diet, BH4, L-dopa/carbidopa,5-OH-tyrptophan, folinic acid
Pterin-4-carbinolamine-dehydratase deficiency	Liver, kidney, intestine	10q22	Tetrahydrobiopterin Deficiency	Hyperphenylalaninemia, mild tremor, neonatal hypotonia, high neopterin, subnormal biopterin, Primapterin is present	Low-phe diet, BH4

History

Dr. A. Foelling, a Norwegian physician discovered PKU in 1934 [8]. He added ferric chloride to the urine of two children with intellectual disability and observed the deep green color change in their urine sample. He then used chemical analysis to determine the nature of the substance that caused that deep green color change and extracted phenylpyruvic acid from their urine. About 20 years later, a German professor, Horst Bickel, developed the first Phenylalanine- free formula that could decrease blood phenylalanine levels and prevent intellectual disability [9]. In 1962, Robert Guthrie developed a bacterial inhibition assay to measure phenylalanine level on a filter paper [10]. The test was fast, simple and inexpensive, and had acceptable specificity and sensitivity. Newborn screening for PKU was first started in Massachusetts in 1963. It was very successful, and many other states mandated the screening by the mid-1960s in the USA. Today, PKU is screened in all states of the USA and many other countries in all over the world allowing early diagnosis and better health outcome as well as resulting in less economic burden on the health system.

Diagnosis

Newborn screening

Phenylalanine levels are measured with tandem mass spectrometry (MS/MS) using dried blood spots in all newborns in US and many developed countries as early as 24 hours of life. Many laboratories also calculate and report the phenylalanine and tyrosine ratio to increase the specificity of the screening test. Newborns with Phe levels higher than 2 mg/dL (120 μmol/L) are reported as a positive screen. Elevated phenylalanine level can be caused not only by PKU but also defects of tetrahydrobiopterin, prematurity, liver disease and high protein intake. Elevated phenylalanine (Phe):tyrosine (Tyr) ratio over 2.5 mg/dL increases the likelihood of PKU [11]. A mean cutoff for Phe of 130 μmol/L (with a range of 65–234 μmol/L) and a Phe:Tyr ratio > 3 is abnormal [12]. A metabolic physician should see newborns with elevated phenylalanine levels as soon as possible. Blood amino acid quantification should be performed as a diagnostic follow-up of positive newborn screen. Elevated phenylalanine level and normal levels of other amino acids in plasma amino acid (PAA) analysis confirm the diagnosis of PKU. Further guidance on follow-up of an abnormal NBS test for Phe can be found in the ACMG ACT sheets for newborns along with confirmatory testing algorithms (www.acmg.net).

PAH gene sequencing is a clinically available test and also confirms the diagnosis of PKU.

Cofactor deficiency testing

It is essential to measure urine pterins and dihydropteridine reductase (DHPR) enzyme activity on a filter paper to rule out defect of tetrahydrobiopterin (BH₄) synthesis and regeneration in all newborns with elevated Phe. If that will result in delay of diagnosis, then a 24-hour BH₄ loading test can be performed in addition to the urine pterins and DHPR enzyme activity. The blood and urine samples should be collected before BH₄ loading dose.

PAH enzyme activity

PAH enzymatic activity is detectable in hepatic and renal tissues only and is not appropriate for either screening or diagnostic testing. The diagnosis of PKU is established when the Phe concentration is above 120 μmol/L with altered Phe to tyrosine ratio and normal BH₄ cofactor metabolism and/or finding biallelic pathogenic variants in PAH.

Genetics

The human PAH gene was mapped on chromosome 12 q22–q24.1 and contains 13 exons and 12 introns (Table 1). About 1,184 PAH variants (PAHvdb database; http://www.biopku.org; last accessed 06/22/2020) are known to be associated with PAH deficiency. The majority of the reported variants (60%) are missense, but deletions, duplications, and insertions are also observed. Missense variants usually result in abnormal folding of the PAH protein, increased protein turnover, decreased activity and/or impairment of catalytic functions. The genotype is clearly the best clinically available predictor of severity in PAH deficiency.

Genotyping patients with elevated Phe is not essential for the diagnosis of PKU but it may determine the degree of protein dysfunction, residual PAH activity and consequently the metabolic phenotype and therapy planning. Genotyping would help in determining the extent of dietary Phe restriction and the likelihood of response to BH₄ supplementation. Alleles that are known to be responsive to treatment with BH₄ are listed in the BIOPKU database. http://www.biopku.org/home/biopku.asp.

p.Arg408Trp (c.1222 C > T) in exon 12 is the most common pathogenic variant in Europe [13]. IVS12 + 1G>A (c.1315 + 1G>A) at the splice donor site of intron 12 is a common pathogenic variant in northern European ancestry. IVS10–11G>A is a splice site of intron 9, is the second most frequently reported pathogenic variant in PAHvdb and prevalent in southern Europe/Mediterranean populations and associated with the most severe phenotype [14].

While large numbers of variants have been reported as BH₄ responsive, there is inconsistency in BH₄ responsiveness in patients with the same variants [15].

Genetic counseling

PAH deficiency is inherited in an autosomal-recessive manner; in which the affected individual will inherit two copies of the pathogenic variants in the PAH gene, one from each parent. At each pregnancy, there is a 25% chance of having an affected child, a 50% of asymptomatic carrier, and a 25% chance of having unaffected and not a carrier child. Carrier testing for at-risk relatives and prenatal testing for pregnancies at increased risk are available if the PAH pathogenic variants have been identified in an affected family member.

Pathology

In morphologic descriptions of untreated PKU, both gray and white matter changes are noted [16, 17]. Minor architectural abnormalities of the cortex are described, such as lobar disproportion and gyral convolutional irregularities. The major changes are in the white matter, where vacuolation is seen in the central nervous system (CNS) of children and demyelination and astrogliosis in the CNS of adults. Vacuolation consistently involves the central white matter of the cerebrum and cerebellum, optic tracts and fornix. Demyelination follows the same pattern with a tendency for preservation of articulate fibers. Intense astrogliosis and sudanophylic and periodic acid-Schiff (PAS) - positive macrophages are also evident. One ultra-structural study describes membrane bound; parallel lamellar inclusions believed to be within oligodendrocytes [18]. Lipids and proteolipids associated with myelin are decreased [19]. A review of histopathology and neuroimaging studies shows that diffuse white matter pathology in untreated PKU patients likely reflects hypomyelination, while in early treated patients white matter abnormalities observed on magnetic resonance imaging (MRI) likely reflects intramyelinic edema [13].

The relationship between intellectual disability and morphologic and biochemical alterations is not clear. Some data points to impaired protein synthesis, perhaps because high blood levels of phenylalanine inhibit cerebral uptake of amino acids that share the same transporter with phenylalanine. Other data suggest a deficiency of brain dopamine, since this neurotransmitter forms from tyrosine that, in turn, derives from phenylalanine in the reaction (phenylalanine hydroxylase) that is defective in PKU [20, 21].

Evidence also indicates altered energy metabolism in the brain of the child with PKU [22–24]. Phenylalanine and its metabolites appear to impact several aspects of brain energetics including: (a) inhibition of glucose uptake; (b) diminished glycosylation of cytoskeletal proteins; (c) inhibition of hexokinase and pyruvate kinase; (d) reduced flux through the glycolysis and (e) inhibition of mitochondrial electron transport chain.

Clinical findings

PKU can cause severe intellectual disability that is preventable by the institution of a protein-restricted diet in early infancy [1, 25, 26]. Patients with untreated PKU appear normal in the first few months of life and then it is noticed that they fail to achieve developmental milestones in time and their head circumference does not grow [1]. Most untreated patients come to medical attention after 6–9 months of life because of developmental delay, musty body odor, microcephaly and seizures. Their complexion is usually much lighter than their parents and siblings secondary to the associated inhibition of tyrosinase and low tyrosine levels. Eczema occurs in about 30% of patients. In older untreated children, severe intellectual disability, epilepsy, aggressive behavior, self-mutilation, autistic features, schizophrenia-like symptoms, Parkinson-like features are common [1] (Table 2).

Table 2

Symptoms of untreated Classic PKU

Symptoms
Odor (urine and body)
Lighter pigmentation in family constellation
Intellectual disability
Microcephaly
Irritability
Seizures
Hypertonia
Autism
Self mutilation
Rash, eczema

Fortunately, early diagnosis and treatment through newborn screening has changed the clinical phenotype of PKU in many developed countries, allowing them to grow up with normal intelligence, however, lower than that in sib controls [27–31]. Implementation of a Phe-restricted diet shortly after birth significantly reduces blood Phe levels and prevents many of the neurologic manifestations of PKU [1, 25]. Lifelong adherence to a low phenylalanine diet is extremely difficult [32, 33]. If patients maintain good control as children and go off diet as teenagers or adults, significant difficulties with executive function concentration, attention deficit, emotional lability, anxiety and depression may occur [27, 29, 34]. Executive dysfunction may also occur in early treated children in spite of diet treatment [27, 34]. The severity of psychiatric symptoms correlates with elevation in blood Phe level, and reduction of Phe level generally results in symptom improvement. Given the increased risk for neurocognitive and psychological issues in patients with PAH deficiency, regular mental health monitoring is warranted [25].

It is important to closely monitor the bone health of individuals with PAH deficiency given the risk of osteopenia and low Z-scores [35].

Differential diagnosis

The differential diagnosis of hyperphenylalaninemia (HPA) includes high natural protein intake, prematurity, defects in BH₄ metabolism and liver disease. Patients with disorders of BH₄ metabolism including GTP cyclohydrolyase I (GTPCH) deficiency, 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, dihydropteridine reductase (DHPR) deficiency and pterin-4a-carbinolamine dehydratase (PCD) deficiency can present with any degree of HPA [36, 37].

Treatment

Dietary therapy

Treatment should be started immediately upon diagnosis to prevent neurological damage and it must be life long, with a goal of maintaining Phe in the range of 120–360 μmol/L. It was found that a 4-week delay in starting treatment caused a decline in IQ score by 4 points [38]. Based on the initial blood Phe levels, Phe may be excluded from the diet until levels approach the treatment range followed by initiation and titration of a Phe-restricted diet. The mainstay of treatment of PKU is a low–phenylalanine diet. The general consensus is to start diet treatment immediately in patients with Phe levels above 600 μmol/L [25]. Patients with blood Phe level <360 μmol/L should remain untreated, however, they will need to be monitored closely, especially in the first 2 years of life as there is a possibility of increase in the blood Phe level with age with protein intake [39, 40]. If treatment is not required before 2 years of age, then those patients need to be monitored on an annual or biennial basis.

Patients with Phe level between 360 and 600 μmol/L should be treated at least during the first 12 years of life to prevent cognitive impairment [41]. This is particularly important in women given the risks associated with maternal PKU and Phe level >360 μmol/L. However, there is no clear evidence of treating patients with Phe level between 360 and 600 μmol/L as previous studies showed mixed clinical outcomes of untreated patients ranging from normal cognition to subtle neurocognitive deficits [42–45].

Most physicians also advocate phenylalanine-restricted diet in patients with mild hyperphenylalaninemia whose levels are persistently above 360 μmol/L. The basic principle of the diet is to restrict the intake of phenylalanine while providing phenylalanine-free protein. This is achieved by replacing a certain quantity of phenylalanine containing foods (the amount replaced is adjusted according to each patient’s tolerance) with synthetic, phenylalanine – free formula and medical food to provide all other nutrients recommended for the patient’s age and necessary for normal growth and health maintenance [46]. A wide range of Phe-free formulas and low protein medical foods such as low protein-pastas and bread are available today to improve the compliance with the dietary Phe-restriction.

At the start of diet treatment, patients with phenylalanine levels above 800 μmol/L require phenylalanine free formula for 12–48 hours to bring their phenylalanine levels close to 360–420 μmol/L. To achieve smooth control, phenylalanine should be added once the phenylalanine level approaches the upper end of therapeutic range (about 400 μmol/L). Small changes in dietary phenylalanine allowance should be made based on blood phenylalanine levels that are monitored closely, ideally, every day in the first couple of days of treatment [46, 47]. It is important to have consistent testing methodology for better comparison over time. Long-term management, serial monitoring of Phe levels is essential to achieve good metabolic control. Although current best practices concerning the frequency of monitoring varies from one center to another, it is usually recommended once a week in the first 2 years, every other week by school age, once a month after 7 years of age [25].

In the early years of life, especially during rapid growth, infants with classical PKU may tolerate 300–400 mg/day phenylalanine; during linear growth, such children usually tolerate 200 mg/day Phe to keep their Phe levels within therapeutic range. There is no consensus regarding optimal levels of blood Phe across different countries and among treatment centers in a single country. In the USA, the most commonly advocated therapeutic range is 120 to 360 μmol/L (2 to 6 mg/dL) in the first 12 years and then 120–600 μmol/L (2–10 mg/dL) [25].

Dieticians should carefully follow patients and their 3-day-diet records should be reviewed regularly to prevent nutritional deficiencies. For further detailed dietary management of PAH deficiency, please refer to the nutrition guidelines by the Genetic Metabolic Dietitians International (www.gmdi.org) and Southeast Regional Genetics Network (www.southeastgenetics.org). Based on nutritional assessment and concern of suboptimal dietary intake, additional monitoring blood work should include PAA, transthyretin, albumin, complete blood count, ferritin, 25-OH vitamin D, vitamin B12, red blood cell essential fatty acids, trace minerals (zinc, copper, and selenium), vitamin A, comprehensive metabolic panel, and folic acid. Additionally, bone health should be considered given the suboptimal bone mineralization secondary to the low intake of calcium from the protein-restricted diet. However, the utility of routine DEXA scans to monitor bone density is controversial [35]. Additionally, adolescent with PKU are at risk of vitamin B12 deficiency if they are less adherent to their diet and amino acid supplementation and continue to choose low protein diet [48].

Although it is very well known that PKU patients should maintain a low protein diet for life, several factors can make it difficult to follow: many dislike the taste of the low-Phe medical food; it is costly and sometimes not covered by health insurance; many feel strong peer pressure to eat a normal diet [47].

Pharmacotherapy

Large neutral amino acids

Administration of large neutral amino acids (LNAA) has been introduced as another approach to diet therapy. LNAAs (tyrosine, tryptophan, arginine, leucine, isoleucine, valine, methionine, histidine, lysine, threonine and phenylalanine) use the same large neutral amino acid type 1(LAT-1) transporter to cross the intestinal cell membrane and blood brain barrier (BBB) [49, 50]. Binding of LNAA to the transporter is a competitive process. LAT- 1 transporter has different affinities and K_m values for each LNAA, and phenylalanine has the lowest K_m value indicating that it binds to the transporter more strongly than other LNAAs. In PKU, the phenylalanine level is much higher than that of other LNAAs, and phenylalanine readily crosses intestinal cell membrane and blood brain barrier and inhibits the transport of other LNAAs. The rationale for use of LNAA is that these molecules compete with phenylalanine for transporters across the BBB; therefore, large concentrations of other LNAAs in the blood reduce the uptake of phenylalanine into the brain. In addition to LNAAs blocking the influx of excess phenylalanine into the brain in PKU, LNAA may also promote protein synthesis [51].

The brain concentration of phenylalanine decreases with LNAA tablets despite increased natural protein intake [51]. Pietz at al reported that the LNAA blocked the phenylalanine transport to the brain by giving an oral phenylalanine challenge with or without LNAA and measuring the influx of phenylalanine into the brain [52]. Matalon et al. in 2006, showed about 40% reduction of blood Phe following substitution of a standard low Phe medical food with one supplemented with LNAA at a dose of 0.5 or 1.0 g/kg of body weight [53]. The use of the LNAA therapy has been shown to improve amino acids profiles as well as increase tyrosine and tryptophan levels in the blood, which are precursors for dopamine and serotonin [54].

LNAA are not offered to children or pregnant women due to a limited understanding of their effects on fetal growth and the developing central nervous system of the fetus and children but are considered appropriate for treating adults over 18 years of age, adolescents with poor dietary adherence and those with late diagnosed/treated PKUs. It is recommended that LNAA comprise 25–30% of a patient’s daily protein requirement and the remaining 70–75% comes from natural proteins [54].

Glycomacropeptide

Glycomacropeptide (GMP), a natural by-product of cheese that has very low Phe content, has been used in several medical foods as another approach of improving the nutritional management of PKU [55]. GMP is the only known dietary protein that is naturally free of phenylalanine in its pure form. GMP can be used as an alternative to amino acid formula as a source of low-phenylalanine protein for the PKU diet. However, there are some drawbacks to GMP. Isolation of GMP from cheese whey results in contamination with other whey proteins that contain phenylalanine and other amino acids not found in pure GMP. Commercially available GMP contains 2.5–5 mg phenylalanine/g of protein. GMP requires supplementation with amino acids to provide complete source of protein for PKU patients [56].

Sapropterin

In light of the challenges that many patients face in complying with the ongoing demands of a phenylalanine-restricted diet, a prescription medication that would permit better control of phenylalanine without the need for severe dietary restriction would significantly contribute to the management of PKU. Accordingly, researchers have sought alternate approaches to therapy. One strategy is the use of tetrahydrobiopterin (BH₄) to enhance activity of pterin-dependent amino acid hydroxylases, one of which is phenylalanine hydroxylase, the others being tyrosine hydroxylase, and tryptophan hydroxylase. Each enzyme catalyzes the hydroxylation of the aromatic side chain of its respective amino acid substrate using molecular oxygen and a BH₄ as substrates.

In 1975, Milstien and Kaufman suggested that BH₄ might stimulate PAH in patients with residual activity [57]. Kure et al. confirmed this hypothesis in 4/5 patients with mild PKU in whom BH₄ loading reduced blood phenylalanine by 40–70% while patients consumed a normal diet [58]. Since then, numerous studies have confirmed this finding [59–71]. Even patients with classic PKU occasionally respond to the point that it becomes possible to ease dietary restrictions or even replace the diet with a near-normal regimen. In December 2007, the FDA approved Sapropterin dihydrochloride (Kuvan), a synthetic form of the naturally occurring cofactor, tetrahydrobiopterin, at a recommended dose of 10 to 20 mg/kg/day. About 25–50% of the patients with PAH deficiency are sapropterin-responsive. In general, this treatment is considered successful when patients respond with at least a 30% decrease in phenylalanine level with the assumption that the diet remains stable throughout the testing period. The responsiveness is assessed based on frequent measurements of blood Phe level just before starting the sapropterin as a baseline, then obtaining follow up Phe level at 24 hours, 1 week, 2 weeks and sometimes 3 and 4 weeks. The recommended sapropterin testing dose is not less than 20 mg/Kg as it could underestimate the response rate. Sometimes, there is a delay in the effect of sapropterin on blood Phe up to 2–4 weeks. If there is no actual decrease in blood Phe level, but there is improvement in neuropsychiatric symptoms or increase in Phe tolerance, that should justify the sapropterin therapy as well. However, assessing the success of Kuvan with infants and small children on diet therapy requires more sensitive measurement. Such infants and children may be defined as responders to Kuvan if they can double their phenylalanine intake while keeping their Phe levels within the recommended range. For such children, Kuvan offers the opportunity to tolerate a less restricted diet allowing them to eat more natural protein while maintaining their blood Phe level in the desired range. Some patients could actually double and even triple their dietary Phe intake resulting in much better quality of life. Long-term therapy with sapropterin in responsive patients should be maintained lifelong. As mentioned previously, genotyping would help in predicting the response to BH₄ supplementations. However, it is still difficult to predict the phenotype based on the genotype. So, the current guidelines recommend that every PAH-deficient patient should be offered a trial of sapropterin therapy to assess responsiveness except those with two pathogenic null variants in trans [25].

Enzyme substitution therapy

Polyethyleneglycol-conjugated phenylalanine ammonia lyase (PEG-PAL) or Pegvaliase is an FDA approved PAH enzyme substitution since May 2018 [25, 72]. It showed to be effective regardless of residual enzyme activity and has been only FDA- approved for use in adults who have uncontrolled blood Phe concentrations >600 μmol/L [73]. Phenylalanine ammonia lyase is a bacterial enzyme that degrades Phe to ammonia and trans-cinnamic acid; ammonia is metabolized by the liver while trans-cinnamic acid and its final product, benzoic acid, are conjugated with glycine and excreted in the urine [74, 75]. Pegvaliase therapy aims to provide maintenance of blood Phe concentrations while normalizing the diet in all adult patients with PKU who can adhere to the therapy and able to monitor for potential adverse events [73, 76].

Results of a phase III PRISM program support the efficacy of Pegvaliase for the treatment of adults with PKU regardless of residual enzyme activity, with a manageable safety profile in most participants [25, 77, 78]. It showed substantial reduction in blood Phe and improvement in inattention and mood symptoms, however serious adverse events have been reported such as anaphylaxis. The most common adverse events were arthralgia (70.5%), injection site reactions (62.1%), injection site erythema (47.9%), and headache (47.1%) [77, 78]. Therefore, premedication with antihistamine and/or antipyretics is recommended prior to administration of Pegvaliase as well as monitoring the patient during and at least 60 minutes following the administration of the treatment. Given the risk of anaphylaxis, it is important to educate the patients about the signs and symptoms as well as administration of epinephrine if needed [73].

It is recommended to obtain baseline blood Phe concentrations prior to the initiation of Pegvaliase [73]. The induction dosage for pegvaliase is 2.5 mg subcutaneously (SC) once weekly for 4 weeks under the supervision of a health care provider. The dosage then are titrated based on tolerability over at least 5 weeks to achieve a dosage of 20 mg SC once daily until the patient is titrated to an effective maintenance dose where therapeutic response is achieved. Therapeutic response is defined as 20% reduction in blood Phe concentration from baseline or blood Phe concentration concentration ≤600 μmol/L after 24 weeks. If this was not achieved, then consider increasing the Pegvaliase dose to a maximum dosage of 40 mg SC daily. If therapeutic response has not been achieved after 16 weeks on the maximum dosage, discontinuation of pegvaliase is recommended. However, if the patient develops very low blood Phe concentrations (<30 μmol/L) during titration and maintenance, the dosage of Pegvaliase may be reduced or the dietary protein and Phe intake may be adjusted [73]. Pegvaliase can be self-administered if patient shows adequate competency. Of note, the efficacy of pegvaliase without dietary therapy was not assessed in clinical trials and thus it is important to continue monitoring the Phe intake to avoid changing in the dosage of Pegvaliase [78]. Further, it is unclear whether the combination of sapropterin with Pegvaliase is more beneficial than either therapy alone as there are no clinical trials addressing their combined safety and efficacy [78].

Emerging therapies

Gene therapies

Both liver-directed and skeletal muscle directed gene therapy are being investigated in PKU murine models with good success [79–82]. There is an ongoing phase I/II clinical trial, which uses human hematopoietic stem cell-derived adeno-associated virus vectors (AAVHSCs) to deliver a functional copy of the PAH gene to the liver cells [83].

Cell-directed therapies

Liver repopulation with PAH-expressing cells has been investigated by Harding in 2006 to replace PAH-deficient hepatocytes of the affected individual with PAH-positive cells from some other source [82]. It could be a viable approach to the treatment of PKU if a selective growth advantage could be achieved for donor hepatocytes.

PKU treatment for life

The current recommendation is that the treatment should be lifelong for all patient with PAH deficiency maintaining Phe level between 120–360 μmol/L. Liberalization of the Phe-restricted diet and relaxation of Phe control is no longer acceptable practice given the known adverse neurocognitive and psychiatric outcomes that might develop later in life, which include deficits in executive functioning and psychiatric symptoms such as anxiety, depression, and phobias. This will ultimately lead to lower level of educational attainment and socioeconomic status. Additionally, it is very challenging to return to Phe-restricted diet after patients have tried high protein food and found it more palatable [25].

Surveillance

It is important to obtain regular monitoring of PAA concentrations in individuals with PKU. After Phe stabilization in newly diagnosed infants, it is essential to obtain PAA levels weekly in infants in the first year of life to foster optimal growth during the first year of life. Following the first year of life and until 12 years old, biweekly to monthly sampling may be adequate. In adolescents and adults who are stable and well controlled, blood level monitoring can be monthly. Further, regular assessment of growth and micronutrient needs as well as assessment of developmental progress and screening for mental illness are fundamental at every visit [3, 25].

Maternal PKU

Maternal hyperphenylalaninemia causes an embryopathy/fetopathy that compromises growth and causes congenital malformations, microcephaly, and intellectual disability in the fetus [84] (Table 3). It is a consequence of intrauterine phenylalanine excess derived from the positive transplacental gradient. All girls and women of childbearing age with hyperphenylalaninemia including those with classical PKU and milder forms of hyperphenylalaninemia, should be given reproductive counseling and should be on a treatment regimen with strict dietary phenylalanine restriction before conception and throughout the pregnancy; meticulous treatment may be compatible with a normal outcome for the fetus. Pregnant women without adequate blood Phe level will need intensive intervention, including hospitalization to start strict dietary control [25]. Additionally, frequent blood Phe testing and diet adjustments are required throughout pregnancy as maternal Phe requirements change significantly throughout gestation.

Table 3

Maternal PKU syndrome*

Symptoms	Frequency
Mental retardation	92%
Microcephaly	73%
Low birth weight	40%
Congenital heart disease	12%

*Plasma phenylalanine levels over 20mg/dl.

In a large collaboration study of 575 pregnant women with PKU, fetal outcome was significantly improved with early treatment and maintenance of phenylalanine concentrations of 120 μmol/L to 360 μmol/L (2–6 mg/dL) [85, 86]. Microcephaly, which occurred in almost 27% of offspring, was found only in offspring of women in whom treatment began after the first 8 weeks of pregnancy, i.e., late-treated pregnancies. Similarly, almost all cases of congenital heart disease and significant gastrointestinal abnormalities, including tracheoesophageal fistula, occurred in infants of mothers whose blood Phe concentrations were uncontrolled in the first trimester [26]. Hypoplasia of the left heart, infantile coarctation, and right heart outflow tract lesions were seen most frequently. The frequency of intrauterine growth restriction (IUGR) does not differ from that in the general population if maternal Phe levels are controlled by week 10 of gestation.

The best observed outcomes occur when strict control of maternal blood Phe concentration is instituted before pregnancy or by 8 weeks of gestation at the latest [87]. Elevated blood Phe levels in the first 8–10 weeks of gestation are associated with increased risk of congenital heart disease and poor fetal growth [25]. The currently recommended Phe concentrations during pregnancy are (120–360 μmol/L) [2–6 mg/dL] in the United States [87]. There is a linear relationship between maternal Phe levels >360 μmol/L throughout gestation and lower IQ of developing fetus. Maintaining maternal Phe levels <360 μmol/L prior to conception is recommended [25]. However, persistently low maternal Phe level (as low as 100 μmol/L) may be associated with increased risk of IUGR. Additionally, dietary over-restriction leading to inadequate protein and calorie intake, should be avoided as it may increase maternal Phe levels.

Some of the medications and dietary supplements used in PKU patients should not be used during pregnancy, this includes LNAA as they do not consistently affect maternal blood Phe. Sapropterin, which is a class C medication, can be used during pregnancy when the benefits of using it outweigh the risks. There is no evidence of sapropterin-associated teratogenicity; on the contrary, there are many reports of uneventful pregnancy outcomes with its use. Per the ACMG practice guidelines of PKU, women who are on sapropterin and become pregnant, should be offered the option of remaining on the medication and women who are not on it and may benefit from its use, should be offered the option of starting it.

Careful metabolic and nutritional monitoring is required in postpartum period as the Phe requirements decrease. Mothers should continue their Phe-restricted medical food to provide the required calories and protein to support breastfeeding (640 kcal/day and 25 g protein/day). Mothers with PAH deficiency can safely breastfeed [25].

Tetrahydrobiopterin (BH₄) defects

Defects in the synthesis and recycle of the cofactor tetrahydrobiopterin (BH₄) are responsible for a small fraction of cases with hyperphenylalaninemia, approximately 1–2% [1, 88]. Such cases are sometimes called “malignant” PKU because of the progressive deterioration in neurological function, which cannot be treated by a phenylalanine-restricted diet. These cases should be distinguished from PKU caused by PAH deficiency. BH₄ defects cause decreased production of the neurotransmitters dopamine and serotonin. They also result in hyperphenylalaninemia with two exceptions: the autosomal dominant, GTP cyclohydrolase I deficiency (GTPCH), and sepiapterin reductase (SR) deficiency. BH₄ metabolic enzymes include three synthetic enzymes: GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR)-and two recycle enzymes: pterin-4cE-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) (Fig. 2). Despite the low incidence of BH₄ defects, all newborns with hyperphenylalaninemia detected through newborn screening should be screened for BH₄ defects. With the exception of DHPR deficiency, which can be detected by determination of DHPR activity in dried blood spots (DBS), all other forms of BH₄ deficiency (GTPCH, PTPS, and PCD deficiency) can be detected by specific pterin patterns in urine or DBS [36, 37, 89]. Prognosis and outcome strongly depend on the age when the diagnosis is made and treatment introduced, but also on the type of the enzyme defect and pathogenic variant [88, 90].

Fig.2

The Metabolic Pathway of Tetrahydrobiopterin (BH4).

Early diagnosis of GTPCH, PTPS and DHPR deficiencies may prevent irreversible brain damage by pharmacological treatment. Those with PCD deficiency may be at risk of developing non-immune MODY-like diabetes or hypomagnesaemia and renal magnesium wasting [91, 92]. Evaluation for BH₄ disorders for any neonate or infant with neurological problems of unknown origin is suggested even without increased Phe or negative NBS for increased Phe.

GTP cyclohydrolase (GTPCH) I deficiency

Deficiency of GTP cyclohydrolase I can occur in a recessive and dominant form (Table 1). The dominant form, with mutation in only one of the two alleles for GTP cyclohydrolase I, causes dopa-responsive dystonia [88]. Patients with the recessive form have mutations in both alleles for GTP cyclohydrolase I and are usually detected because of elevated phenylalanine on newborn screening and develop neurological dysfunction with axial hypotonia, hypertonia of the extremities, abnormal movements, tremors, seizures, and sometimes autonomic dysfunction [93, 94].

The GCH1 gene is composed of 6 exons on 14q22.1–22.2. More than one hundred different mutations have been identified in patients with different forms of GTP cyclohydrolase I deficiency [95]. Only 7 out of 110 mutant alleles are present in a homozygous or compound heterozygous state, causing the autosomal recessive form of GTP cyclohydrolase I deficiency [95]. All the others are present at the heterozygous state and cause dopa-responsive dystonia.

Autosomal dominant form of GTP cyclohydrolase 1-deficiency (dopa-responsive dystonia or Segawa syndrome) usually presents with gait disturbance caused by foot dystonia between ages one and 12 years and progresses to Parkinsonism [96]. Diagnosis is confirmed based on biochemical findings, response to oral administration of levodopa (L-dopa) and molecular testing. Concentrations of total biopterin and total neopterin in cerebrospinal fluid (CSF) are reduced in individuals with GTPCH1 deficiencies [96]. Patients are treated with levodopa/decarboxylase inhibitor (initial recommended dose ≤25 mg/day in children, 50 mg 1×–2×/day in adults). The dose should be gradually increased as needed.

AR form of GTP cyclohydrolase 1-deficiency presents with complex neurological dysfunction, including intellectual disability, global developmental delay, limb spasticity, truncal hypotonia, tremors, oculogyric episodes and seizures. While most patients have elevated plasma phenylalanine levels (300 and 1200 μmol/L) in the neonatal period, cases with normal phenylalanine levels also have been reported [93]. Diagnosis is based on elevated phenylalanine levels, decreased total biopterin and total neopterin levels in urine, and CSF. Molecular genetic testing can also help to confirm the diagnosis. The principles of treatment are low Phe diet, BH₄ supplementation (5–10 mg/kg/day), and administration of L-dopa/carbidopa (5–10 mg/kg/day) and 5- hydroxy-tryptophan to improve neurotransmitter deficiency.

6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency

PTPS, an autosomal recessive disorder, is the most common BH₄ defect causing BH₄ deficiency [88]. PTPS causes not only hyperphenylalaninemia but also deficiency of dopamine and serotonin because of malfunctioning BH₄-dependent tyrosine and tryptophan hydroxylases (Fig. 2). PTPS should be suspected in all newborns with elevated phenylalanine levels detected through newborn screening, especially if phenylalanine is moderately elevated [94]. The characteristic findings are intellectual disability, hypo or hypertonia, seizures, irritability, abnormal movements, recurrent hyperthermia, hypersalivation, failure to thrive, microcephaly and difficulty swallowing (Table 1) [97–99]. Symptoms are usually present at birth but become evident by 4–5 months of age. Brain MRI shows early brain atrophy [98]. Measuring pteridin levels in urine can make the diagnosis. 5-hydroxyindolacetic acid (5-HIAA) and homovanillic acid (HVA) levels are low in CSF. BH₄ loading (20 mg/kg), which shows prompt decrease in phenylalanine level after BH₄ supplementation, is commonly used as a diagnostic tool in Europe. Urine pteridin profile is characteristic for PTPS deficiency. There is decreased or undetectable biopterin and markedly elevated neopterin. RBC PTPS activity can also be measured and is very low in most typical patients.

The mainstay of treatment is to decrease phenylalanine level by protein restricted diet or BH₄ supplementation (5–10 mg/kg/day), and to restore neurotransmitter deficiency by giving L-dopa/carbidopa (5–10 mg/kg/day) and 5- hydroxy-tryptophan (5–10 mg/kg/day). The doses of medications should be adjusted based on response to therapy, clinical improvement as well as adverse effects for each individual patient [98, 99]. There are no good biomarkers to measure the response to treatment except prolactin level [100]. Hyperprolactinemia is a good indicator of hypothalamic dopamine deficiency. Measuring neurotransmitter levels in CSF is a direct but not practical way to evaluate the response.

Sepiapterin reductase (SR) deficiency

SR deficiency, an autosomal recessively inherited disease, is the most recently discovered inherited defect in the synthesis of BH₄. SR can be difficult to diagnose because patients have normal plasma phenylalanine levels and cannot be detected through newborn screening [94]. The SPR gene is located on chromosome 2p14–p22 (Table 1). The characteristic features are intellectual disability, dystonia with diurnal fluctuations, oculogyric crises, axial hypotonia, and spasticity [101, 102]. Diagnosis of SR deficiency requires CSF studies, which indicate the presence of sepiapterin as well as low concentrations of homovanillic acid, 5-hydroxyindol- acetic acid. It is best to supplement CSF studies with enzyme and genetic analyses. A marked decrease of sepiapterin reductase activity of the fibroblasts along with sepiapterin reductase gene (SPR gene) molecular analysis confirm the diagnosis [103].

The treatment is to administer L-dopa/carbidopa (1–10 mg/kg/day in 3–4 doses) and 5-hydroxytrptophan (1–8 mg/kg/day in 3–4 doses). The dose should be adjusted based on clinical response and adverse effects. Levodopa/carbidopa/5- hydroxy-tryptophan therapy may reduce cerebrospinal fluid folates (CH3-group trapping by levodopa to 3-metoxy- dopa) requiring folinic acid substitution (15 mg/day). Patients may also need BH₄ supplementation [104].

Pterin-4cE-carbinolamine dehydratase (PCD) deficiency

PCD deficiency, an autosomal recessive disorder, has milder phenotype compared to other BH₄ defects (Table 1) [90]. Patients have elevated phenylalanine levels that can be detected through newborn screening [94]. Neurodevelopment is good if phenylalanine levels are maintained within acceptable range. Some patients develop mild upper extremity tremors after stimulation. Hypotonia and irritability has been reported in the newborn period. In PCD deficiency neopterin is initially high, biopterin is in the subnormal range and primapterin is present in urine. Low phenylalanine diet and/or BH₄ therapy (10 mg/kg/day) is recommended treatment for this disorder.

Dihydropteridine reductase (DHPR) deficiency

DHPR deficiency is an autosomal recessive disorder in the regeneration pathway of tetrahydrobiopterin (BH₄). Individuals with DHPR deficiency present with hyperphenylalaninemia, microcephaly, hypotonia, intellectual disability and convulsions [90, 105] (Table 1). DHPR is encoded by the gene QPDR. Missense variants, deletions/insertions and splice site variants in the QPDR gene have been associated with DHPR deficiency [106–108]. Patients have elevated phenylalanine levels and can be detected through newborn screening [94]. DHPR enzyme activity can be measured on dry blood spot. Urine pteridin test shows very high biopterin levels.

Although there is no effective treatment for DHPR deficiency, low protein diet, L-dopa, and 5-hydroxytryptophan supplementations are the mainstay of treatment to keep phenylalanine levels within normal range and increase levels of neurotransmitters. Folinic acid supplementation is also utilized as DHPR is associated with the maintenance of appropriate folate levels and patients with DHPR deficiency have low folate (5-Methyltetrahydrofolate) levels in CSF studies [109]. BH₄ supplementation is not routinely recommended because there is a concern that BH₄ therapy may further increase BH₂ production which will cause decreased BH₄/BH₂ ratio in patients with DHPR deficiency. In one case we showed that BH₄ supplementation improved the clinical symptoms and normalized plasma phenylalanine level and no increase in BH₂ level was found in CSF [110].

TYROSINEMIA

Tyrosine is derived either from dietary protein intake, tissue protein breakdown or hydroxylation of phenylalanine. Impaired degradation of the amino acid tyrosine is a feature of several acquired and genetic disorders [111]. Tyrosinemia is indicated by elevated blood levels of tyrosine above 200 μmol/L. Tyrosine is catalyzed by a series of five enzymatic reactions yielding acetoacetate (ketogenic) and fumarate (gluconeogenic). Only liver and renal proximal tubules express the complete pathway and contain all the enzymes required for tyrosine catabolism. The metabolic pathway of tyrosine is shown in Fig. 3. Tyrosine participates in the formation of thyroxin, dopamine, norepinephrine, and epinephrine (Fig. 2). Inborn errors of tyrosine degradation include (Table 4): (1) Tyrosine aminotransferase deficiency (Tyrosinemia type II), (2) 4-hydroxyphenylpyruvate dioxygenase deficiency (Tyrosinemia type III and Hawkinsinuria), (3) Homogentisic acid oxidase deficiency (Alkaptonuria), (4) Fumarylacetoacetase (FAH) deficiency (Tyrosinemia type I). Six patients with Maleylacetoacetate isomerase deficiency (the enzyme preceding FAH in tyrosine degradation) have been recently reported [112].

Fig.3

The Metabolic Pathway of Tyrosine.

Table 4

Inherited disorder of tyrosine metabolism

Enzyme defects	Tissue distribution	Chromosomal localization	Disorder	Characteristic	Laboratory findings	Treatment
Fumarylacetoacetase	Generalized	15q23	Tyrosinemia I	Acute liver failure, hepatomegaly, failure to thrive, vomiting, cirrhosis, renal tubular dysfunction	Succinylacetone in blood and urine, elevated alfa fetoprotein, elevated PT, PTT	Orfadin^® (nitisinone), Tyrosine, Phenylalanine restricted diet
Tyrosine aminotransferase	Liver, kidney	16q22.1–q22.3	Tyrosinemia II	Bilateral pseudodendritic keratitis, painful palmoplantar hyperkeratotic lesions, photophobia, lacrimation, mild learning disability	Elevated plasma tyrosine (>500μmol/L), elevated urine 4-hydroxyphenyl-pyruvic acid, 4-hydroxyphenyllactate, and 4-hydroxyphenylacetate	Tyrosine, Phenylalanine restricted diet
4-OH-phenylpyruvate dioxygenase	Liver, kidney	12q14-qter	Tyrosinemia III	Mental retardation, ataxia	Elevated plasma tyrosine, elevated urine 4-hydroxyphenyl-pyruvic acid, 4-hydroxyphenyllactate, and 4-hydroxyphenylacetate	Low protein diet (?)
4-OH-phenylpyruvate dioxygenase	Liver, kidney	Unknown	Hawkinsinuria	Failure to thrive, transient metabolic acidosis, mild mental retardation	Presence of Hawkinsin in urine organic acid analysis	Low protein diet, vitamin C (?)
Homogentisic acid oxidase	Liver, kidney	3q2	Alkaptonuria	Arthritis, ochronosis, aortic, mitral valve disease, aortic dilatation, myocardial infarction, dark urine	Presence of homogentisic acid	No specific therapy. Potential treatment: Orfadin^® (nitisinone)

Tyrosinemia type I

Tyrosinemia type I, also called hepatorenal tyrosinemia, is an autosomal recessive disorder of tyrosine metabolism, and is caused by deficiency of fumarylacetoacetate hydroxylase (FAH) enzyme (Fig. 3). The accumulation of succinylacetone and related metabolites are responsible for the disease that affects, in particular, liver and kidney function [113–115]. The estimated incidence of tyrosinemia type I is about 1 in 100,000. It is more common among specific ethnic enclaves such as the population in the Saguenay-Lac-St. Jean area of Quebec, where carrier rate and incidence are about 1 in 20 and 1 in 1,846, respectively [116].

Genetics

Fumarylacetoacetate hydroxylase (FAH) gene has been mapped to chromosome 15q23–q25. The gene span is about 35 kb and consists of 14 exons. In 1992, the first pathogenic variant causing tyrosinemia type I was reported [117]. It was a point mutation in which an isoleucine residue substituted for an asparagine at position 16. Many other pathogenic variants subsequently have been reported, and there is high degree of heterogeneity [118]. A variant in the intron 12-donor site (IVS12DSg⁺⁵a) has been found in patients of different ethnic origins (e.g., Iranians, French Canadians and Scandinavians), which indicates an ancient origin and a founder effect. The pathogenic variant was found in 100 % of the patients from Saguenay-Lac-St. Jean area, and 80% of the patients were homozygous for the variant [116].

The genotype data so far does not permit conclusions on genotype-phenotype correlations. Correlations may eventually emerge, but other factors clearly are important because both the acute form and chronic form have been found in patients with the same genotype.

Genetic counseling and prenatal testing

Genetic counseling should be provided to the parents of affected children with tyrosinemia type I as it is inherited in autosomal recessive pattern with 25% recurrence risk with each future pregnancy. This should include discussion of the available molecular testing for confirmation of the diagnosis, testing carriers as well as the possibility of prenatal testing and pre-implantation genetic testing once the FAH pathogenic variants have been identified.

Prenatal biochemical testing is also available by measuring succinylacetone in the amniotic fluid obtained by amniocentesis at 15–18 weeks of gestation. However, false negative results have been reported and thus molecular testing is always preferred for prenatal diagnosis [119].

Pathology and pathophysiology

Patients who are not detected through newborn screening and treated right away will show pathological changes and may suffer much more serious consequences.

Histologic examination usually reveals micronoduler cirrhosis, and quite often, marked bile duct proliferation within portal tracts and fibrotic septa with varying degrees of steatosis, and pseudoacinar or pseudoglandular formations around central canaliculus often containing prominent bile plugs [120–122]. A significant accumulation of iron pigment within Kupffer cells and hepatocytes and giant-cell transformation are also observed [123].

In older children with a more chronic course, the liver is characteristically coarsely nodular and frequently enlarged with mixed micronoduler cirrhosis [124]. Steatosis varies in extent between the various nodules, sometimes even within a single nodule. Fibrous septa vary in width and frequently contain a mild lymphoplasmatic infiltrate and little ductular proliferation. Intralobular cholestasis or inflammation is usually insignificant. A large grayish-white nodule of hepatoma (Fig. 4) can be easily distinguished from the surrounding nodules in the cirrhotic liver. Multiple microscopic foci of hepatocellular carcinoma and multiple areas of nuclear atypia and hyperchromatism qualifying as hepatocellular dysplasia may be present [124].

Fig.4

Gross appearance of liver after resection for transplantation showing macronodular and micronodular cirrhosis with large nodule of hepatocellular carcinoma (left) (Figure 7 in the first book).

Liver cell dysplasia is a premalignant condition in patients who did not receive early diagnosis and treatment and such patients usually need liver transplantation. This is highly suggestive of an underlying powerful carcinogenic influence of some abnormal metabolites [125–128]. In dysplastic liver cells, irregular nuclear profiles with large nucleoli and reduction of cytoplasmic organelles are characteristic ultra-structural features [128].

Renal tubular dysfunction, characterized by Fanconi syndrome and hypophosphatemic rickets, can be a major manifestation of tyrosinemia type I. Typical morphologic changes are nephromegaly with microscopically irregular dilations of the proximal tubules and vacuolation of tubular epithelial cells [129]. Glycogen accumulation in collecting tubules has been reported [130], and nephrocalcinosis is fairly a constant feature. Aneuploidy in the renal tubular epithelial cells and pancreatic islet cells has been described as well as chromosomal breakage and rearrangements [131]. Ultrastructural examination reveals simplification of the epithelial cells, with loss of the brush border, and cytoplasmic vacuolation, especially in the peripheral area.

Hyperplasia and hypertrophy of the islets of Langerhans have been reported [120, 132, 133] and have rarely been associated with chronic hypoglycemia although most patients have normal blood glucose levels [129]. Hyalinization of the islets has also been reported in some patients [134].

There are several infrequent pathological findings in advanced cases. Myocardial hypertrophy is characterized by increased numbers of mitochondria in the myocytes [135]. Obstructive cardiomyopathy in 2 symptomatic cases has been described [136]. Axonal degeneration and secondary demyelination, similar to the changes seen in acute intermittent porphyria, were observed in 3 peripheral nerve specimens analyzed after paralytic crises [137].

In 1993, an unusual observation was made in tyrosinemia type 1 patients whose livers were removed at the time of liver transplantation. As is typical, multiple large and small nodules were found [138]; however, an immunohistological analysis with FAH antibody surprisingly, showed that the livers represented a mosaic of antibody positive and negative hepatic tissue. FAH enzyme activity was present in the cross-reacting material-positive nodules. Molecular analysis has revealed that they were generated by somatic reversion of the disease-causing inherited mutation followed by clonal selection of reverted hepatocytes [139]. This observation demonstrated the strong selective growth advantage of spontaneously reverted FAH positive hepatocytes in this disease.

Tyrosinemia type I provided the first example of spontaneous “gene therapy” in the liver. Interestingly, mutation reversion has been observed not only in compound heterozygotes, in which intragenic mitotic recombination could result in restoration of the enzyme activity, reversions also have been found in patients homozygous for the same mutant allele on both chromosomes. This phenomenon requires precise removal of the single base change mutation responsible for the disease. In most patients the amount of reverted tissue is too low to result in a cure of the disease, but it seems likely that some of the interfamilial variability in the clinical course of tyrosinemia type I may be due to somatic mosaicism. Patients with a significant mass of reverted hepatocytes may have a milder form of the disease.

Tyrosine is not toxic to the liver or kidney, but rather causes only dermatological, ophthalmological and possibly neurodevelopmental problems. What then causes liver and kidney damage in patients with Tyrosinemia type I? Fumarylacetoacetate (FAA), the compound that accumulates in FAH deficiency, is a potent alkylator, causing oxidative damage to the cells in which it is generated by reacting with glutathione and sulfhydryl groups of proteins [126, 140, 141]. Importantly, FAA acts on cell autonomously, directly damaging only the hepatocytes and renal proximal tubules in which it is produced and not adjacent cells. Because of its rapid reactivity, FAA itself is not found in body fluids of patients with tyrosinemia type I. Succinylacetoacetate and succinylacetone derived from the reduction of FAA are the principle metabolites of FAA [141]. These compounds are found systemically and testing for them diagnostically is routine. Succinylacetone (SA) is one of the main culprits in tyrosinemia type I. It interferes with the activity of two hepatic enzymes: Parahydroxyphenylpyruvic acid dioxygenase (p-HPPD), resulting in further increase in blood tyrosine, and porphobilinogen (PBG) synthase or enzyme δ-aminolevulinic acid (δ-ALA) dehydratase, with subsequent increase in δ-aminolevulinic acid (δ-ALA), which may induce acute neurologic (porphyria-like) crises in the patients.

The accumulation of FAA within the hepatocytes results in one of two outcomes; either apoptotic cell death or profound perturbation of gene expression. Recent studies have shown that acute accumulation of FAA triggers apoptosis in both hepatocytes and renal tubular cells [142]. Apoptosis is mediated by caspases 1 and 3 and is associated with cytochrome c release. Glutathione is protective, and its intracellular level can clearly modulate the threshold for FAA-induced apoptosis [142].

Of note, the elevation in blood tyrosine levels seen in other disorders of the tyrosine degradation pathway other than tyrosinemia type I are associated with dermatological, ophthalmological and possible neuro-mental problems, but with no manifestation of liver disease or renal tubular dysfunction. Tyrosinemia types II and III variably respond to phenylalanine and tyrosine-restricted therapies [143–145]. However, such dietary restriction in patients with tyrosinemia type I, even when started early in life, did not prevent the hepatic, renal or neurologic manifestations of the disease.

Clinical findings

Tyrosinemia type I presents either as an acute or chronic form. The acute form presents in the first months of life with acute liver failure, hepatomegaly, failure to thrive and vomiting [111, 113] (Table 5). The chronic form is characterized by chronic liver disease with a high incidence of hepatocellular carcinoma, renal tubular dysfunction with hypophosphatemic rickets, porphyria like episodes, and usually presents in early childhood (Table 5). Patients with the chronic form of the disease have mild hepatomegaly, failure to thrive due to renal tubular dysfunction and a history of easy bruising. All of these complications are proven to be preventable with the initiation of nitisinone treatment.

Table 5

Manifestations of Tyrosinemia Type I

General Clinical	Failure to thrive
Bleeding/bruising
Rickets
Growth retardation in infancy
Liver	Hepatomegaly
Cirrhosis
Jaundice
Ascites
Hepatocellular carcinoma in childhood
Kidney	Tubulopathy
Gastrointestinal Tract	Vomiting
Diarrhea
Central Nervous System	Neurologic symptoms of acute intermittent porphyria (abdominal pain, hypertension, muscular weakness, painful paresthesias or progressive paralysis) in childhood and later

Distinction between acute and chronic forms is not always clear-cut as some children have a stormy course in the first year of life typical of the acute form and subsequently develop a more indolent course compatible with the chronic form. However, children over 2 years of age who are considered to have the chronic form of tyrosinemia are nonetheless at risk for acute life-threatening liver and neurologic crisis [137].

Patients with the acute form usually present after one month of age with overt liver disease that may become evident with rapid increase liver size, ascites, anasarca and hemorrhagic diathesis secondary to loss of synthetic function for clotting factors resulting in marked prolongation of Prothrombin (PT) and partial thromboplastin times (PTT) [111, 146, 147] Unlike most forms of liver disease, the serum transaminase levels are modestly elevated, and bilirubin may be normal of slightly elevated.

Jaundice is usually a terminal event and is rarely observed in infants surviving an acute episode. Infants with liver decompensation emit a typical odor of boiled cabbage or rotten mushrooms. Untreated children rapidly develop cirrhosis with varying degrees of clinical severity. During the first 2 years of life, the child is particularly at risk for episodes of liver decompensation and neurologic crisis [148]. Thereafter, the dominant problems include renal involvement and risk of developing hepatocellular carcinoma. Hepatosplenomegaly occurs in approximately 70% of cases. Rickets may be apparent in those with moderate or severe renal disease [148].

Tyrosinemia type I should be suspected in any infant or child with evidence of hepatocellular disease, cirrhosis for which the cause is not evident [149] (Table 6). Rickets or characteristic renal or neurologic findings, especially if associated with abnormal hepatic function, also suggest this diagnosis.

Table 6

Metabolic Disorders That May Progress to Cirrhosis

Hereditary tyrosinemia*
Galactosemia*
Wolman disease*
Peroxisomal biogenesis defects*
Glycogen storage disease type IV
Neonatal hemochromatosis*
Indian childhood cirrhosis
α₁-antitrypsin deficiency
Wilson disease
Mitochondrial respiratory chain disorders
Familial cholestatic syndromes
Cystic fibrosis
Sialidosis
Gaucher disease
Niemann-Pick disease
Genetic hemochromatosis
*Very rapidly developing

The incidence of hepatocellular carcinoma varies between 17% and 37% [150] in patients who were clinically diagnosed and not treated with Orfadin^® (nitisinone/NTBC). Hepatocellular carcinoma can occur at a very young age and has been reported in a 2.5-year-old child [151]. Metabolic intermediates are natural alkylating agents with characteristics to make them primary candidates for carcinogenesis, and directly cause other cellular damage [126]. Neither CT nor Doppler ultrasound can discriminate between benign and malignant nodules, although the presence of low-attenuation nodules is highly suggestive of hepatocellular carcinoma.

Main findings and complications

Neurologic crisis

There are 2 phases of neurologic crises (1) an active period during which the child experiences painful paresthesias, autonomic signs such as hypertension and tachycardia, similar to acute intermittent porphyria and sometimes progressive paralysis and (2) a period of recuperation, seen in crises with weakness or paralysis. Painful crises are the most frequent neurologic sign, often localized to the legs, and frequently results in a position of extreme hyperextension of the trunk and neck that can be mistaken for opisthotonos or meningismus [111, 148]. Weakness necessitating respiratory support and hyponatremia with associated seizures can also be seen. The neurologic crises are thought to be secondary to axonal degeneration as a result of the demyelination [143].

Recuperation from paralytic crises is possible, although patients with repeated severe crises may have chronic weakness. Vomiting, ileus, and electrolyte imbalance occur frequently, usually prior to the pain crises. The mental development of children with tyrosinemia is normal, and during crises their level of consciousness is not diminished. The active phase of crisis usually lasts for 1 to 7 days.

Neurologic crisis is usually not associated with deterioration of the usual liver function tests. Urinary levels of δ-aminolevulinic acid (δ-ALA) tend to be higher during crisis secondary to inhibition of RBC δ-ALA hydratase, which is believed to explain such pain crises [152–154]. Treatment of the pain crises is mainly through dextrose containing fluids (dextrose 10% with normal saline) at 1.5 to 2 times maintenance rate for age and weight along with analgesics. In case of hypertension and hyponatremia, adequate rapid acting anti-hypertensives and normal saline fluids and sometimes 2% saline are required respectively. NTBC therapy has to be initiated immediately in order to inhibit the production of SA, which are responsible for inhibiting δ-ALA hydratase and resulting in the pain crises. Per the longitudinal study in Quebec in 2012, none of the patients with tyrosinemia type I developed an acute neurologic decompensation while compliant with NTBC therapy [155].

Renal disease

Renal tubular dysfunction and glomerular involvement occur secondary to ongoing presence of succinylacetone (SA) in the blood or urine, confirming the importance of NTBC treatment. Fanconi renal syndrome with generalized aminoaciduria, glycosuria, renal tubular acidosis and hypophosphatemic rickets can occur with normal serum calcium (Ca) [111, 113–118, 120–124, 126–130, 156]. Usually, patients show symptoms of renal involvement by age 6 months. Long-term complications include hypertension, glomerulosclerosis, nephrocalcinosis, and chronic renal failure. Majority of patients with tyrosinemia type 1 have some degree of abnormal renal architecture with dilated tubules, echogenicity, cysts and nephromegaly on ultrasound examination, and may have evidence of mild to moderate nephrocalcinosis. The glomerular filtration rate is generally decreased. Progressive renal disease occurs in untreated patients with tyrosinemia type I. SA is responsible for the renal tubular damage [157]. Soon after diagnosis of tyrosinemia type I, thorough evaluation of renal function should be initiated which include blood testing (BUN, Cr, electrolytes, bicarbonate, calcium, phosphate), urine analysis (glucose, amino acids, calcium, phosphate, Ca/Cr ratio, phosphate, protein, albumin), renal ultrasound, bone X-ray and DEXA scan for evidence of rachitic changes from hypophosphatemia. It is equally important to monitor the renal tubular function periodically, achieved via assessing urine amino acids, glucose, phosphate, protein, albumin or β2-microglobulin, and Ca/Cr ratios compared to plasma [158].

Ophthalmologic consequences

With the NTBC therapy and subsequent improvement in the survival rates in patients with tyrosinemia type I, ophthalmological complications have been raised. Elevated tyrosine levels have been associated with corneal crystals formation resulting in photophobia, lacrimation, pain and redness. The exact threshold of blood tyrosine level associated with corneal crystals is unclear, but the recommendation is to keep tyrosine level be low 600 μmol/L. Corneal crystals associated with photophobia are seen if blood tyrosine concentration is above 600 μmol/L (700–900), which can resolve with improvement in tyrosine level when patients are under strict control of tyrosine intake. The current recommendation is to obtain annual ophthalmologic slit-lamp evaluations patients with tyrosinemia I or when they are symptomatic or at increased risk [158].

Neurobehavioral issues

Neurodevelopmental consequences have come to attention after the improvement in the survival of patients treated with NTBC therapy. There are no studies measuring intelligence in individuals with tyrosinemia type I that were identified through NBS in the United States. However, there are other studies measuring intelligence in tyrosinemia type I patients treated with NTBC, which reported neuropsychological outcomes since the introduction of NTBC treatment. The IQ of patients ranged from 55–115. There is a concern that the hypertyrosinemia produced by the use of NTBC might be associated with long-term behavioral and neuropsychological effects. Accordingly, it is important to include neuropsychological assessment as part of routine follow-up of patients with tyrosinemia type I [158].

Cardiac complications

Hypertrophic cardiomyopathy has been reported in some untreated patients with tyrosinemia type I complicated with chronic liver disease or acute presentation in young infants. The exact mechanism is unclear, but there are different theories attributing it to the inhibition of myocardial porphobilinogen synthase resulting in elevated levels of δ-ALA, which can be cardiotoxic maybe secondary to mitochondrial dysfunction [159]. Further, advanced liver disease resulting in increased cardiac output may also play a role in the pathogenesis of cardiomyopathy. The cardiomyopathy is usually subclinical [160], however there are few patients presented initially with hypertrophic cardiomyopathy before they were diagnosed with tyrosinemia type I estimated as 30% [161]. Cardiomyopathy was significantly less common in those treated initially with NTBC [161]. The typical echocardiogram findings include concentric biventricular hypertrophy and/or interventricular septal hypertrophy. It resolved after treatment with NTBC for about 4 months [162] or liver transplant in severely affected patients.

Endocrine complications

Some may have episodes of hypoglycemia associated with islet-cell hypertrophy [133]. However, most patients are asymptomatic and pancreatic islets cell hyperplasia have been reported as pathologic finding in autopsies. Hypoglycemia could be attributed either to islet cell hyperplasia or to liver dysfunction [158].

Diagnosis

Demonstration of the presence of succinylacetone (SA) on dried filter paper of samples of plasma or urine is pathognomonic [163]. Urine organic acid analysis is an important diagnostic test to measure urine SA as well as tyrosine metabolites such as 4- hydroxyphenyllactate and pyruvate in clinical practice [111, 115, 148] SA and succinylacetoacetate are derived from reduction and subsequent decarboxylation of maleylacetoacetic acid and fumarlyacetoacetic acid. PAA analysis shows elevated levels of tyrosine and methionine. Urine amino acid analysis shows generalized aminoaciduria. Urinary δ-ALA can also be elevated.

The first laboratory indications of liver crisis are disproportionate prolongations of the coagulation time and a bleeding diathesis without other symptoms of liver disease [164]. PT and PTT can be alarmingly prolonged despite normal or near normal serum transaminase levels. Factor V levels, often used in other liver disorders as a marker of liver synthetic function, are usually preserved to within normal or close to normal range. In contrast, factors XI and XII and vitamin K dependent factors II, VII, IX, and X may be exceedingly low with normal factor VIII levels [111]. Highly elevated levels of α-fetoprotein (AFP) are seen, even before the elevation in tyrosine [165].

FAH can be assayed in lymphocytes and erythrocytes as well as on liver tissue. FAH activity is also measurable to some extent in kidney, lymphocytes, erythrocytes, fibroblasts, and chorionic tissue. Carrier detection by these techniques is imperfect, because some heterozygotes for the deficiency can have high levels of residual FAH activity and mosaicism of enzyme in liver.

High concentrations of AFP in umbilical cord blood of affected neonates suggest that liver disease may be present in late gestation.

Elevated levels of methionine and tyrosine in plasma and generalized aminoaciduria, large increase in tyrosine metabolites (4- hydroxyphenyllactic acid, 4- hydroxyphenylpyruvic acid) in urine may also develop in other metabolic disorders associated with hepatocellular disease such galactosemia, hereditary fructose intolerance, fructose 1,6 bisphosphatase deficiency, glycogenosis type IV, and alpha 1 – antitrypsin deficiency.