Classical Phenylketonuria and Other Forms of Hyperphenylalaninemia: Clinical Guidelines

List of Abbreviations

HFA — hyperphenylalaninemia

IUGR — intrauterine growth restriction

MGC — medical genetic counseling

MRI — magnetic resonance imaging

US — ultrasound

PAH — phenylalanine hydroxylase enzyme

PKU — phenylketonuria

Phe — phenylalanine

EEG — electroencephalography

AdGTPCH (autosomal dominant guanosine triphosphate cyclohydrolase) — autosomal dominant guanosine triphosphate cyclohydrolase I deficiency

ArGTPCH (autosomal recessive guanosine triphosphate cyclohydrolase) — autosomal recessive guanosine triphosphate cyclohydrolase I deficiency

BH4 (tetrahydrobiopterin) — cofactor tetrahydrobiopterin

DHPR (dihydropteridine reductase) — dihydropteridine reductase enzyme

GTPCH (guanosine triphosphate cyclohydrolase) — guanosine triphosphate cyclohydrolase enzyme

OMIM (online mendelian inheritance of man) — Online Mendelian Inheritance in Man database

PAH (phenylalanine hydroxylase) — gene encoding the phenylalanine hydroxylase enzyme

PCBD (pterin-4-alpha-carbinolamine dehydratase) — pterin-4-alpha-carbinolamine dehydratase enzyme

PTPS (6-pyruvoyl tetrahydropterin synthase) — 6-pyruvoyl tetrahydropterin synthase enzyme

SR (sepiapterin reductase) — sepiapterin reductase enzyme

µmol/L – micromoles per liter

mg/dL – milligrams per deciliter (1 mg/L equals 60 µmol/L)

^** — the drug is included in the list of vital and essential drugs (Decree of the Government of the Russian Federation No. 2406-r dated October 12, 2019 (Decree of the Government of the Russian Federation No. 2406-r dated October 12, 2019 (as amended on June 9, 2023)).

Terms and Definitions

Hyperphenylalaninemia ― a group of autosomal recessive disorders caused by impaired metabolism of the essential amino acid phenylalanine, which enters the human body with protein-containing foods.

«Maternal phenylketonuria» — embryofetopathy developing in the fetus as a result of exposure to abnormal metabolic products from a pregnant woman with phenylketonuria in the absence of dietary treatment.

Neonatal screening — a medical diagnostic technology of universal unscreened laboratory testing of all newborns for certain hereditary diseases, designed to ensure the timely detection and initiation of treatment in affected children to prevent disability.

1. Brief information on the disease or condition

1.1 Definition of the disease or condition

Hyperphenylalaninemias are a group of autosomal recessive diseases caused by impaired metabolism of the essential amino acid phenylalanine (Phe), which enters the human body with protein-containing foods. Hyperphenylalaninemias (HFA) comprise several genetically heterogeneous forms of Phe metabolic disorders with similar clinical features: phenylketonuria and tetrahydrobiopterin metabolism disorders [2, 16].

Phenylketonuria (PKU) is hyperphenylalaninemia (HFA) caused by deficient activity of phenylalanine hydroxylase (PAH) and leading to the accumulation of Phe and its metabolic products in the body [16, 17, 33].

Tetrahydrobiopterin metabolism disorders (BH₄-deficient HFA) are a heterogeneous group of HFA conditions caused by a deficiency of one of the enzymes involved in the biochemical conversion chain of tetrahydrobiopterin (BH₄) [16, 17, 34, 35].

1.2 Etiology and pathogenesis of the disease or condition (group of diseases or conditions)

Normally in the human body, the bulk of Phe is utilized by conversion into tyrosine, which in turn serves as a substrate for the synthesis of biogenic amines and melanin. Only a small amount of Phe is used for protein synthesis. The conversion of L-phenylalanine to L-tyrosine is carried out by the PAH enzyme (Fig. 1).

Fig. 1. Reaction (1) — conversion of phenylalanine to tyrosine by phenylalanine hydroxylase; reaction (2) — tetrahydrobiopterin (in the presence of Fe2+) is oxidized by dihydropteridine reductase to form dihydropteridin.

The pathogenesis of HFA is based on the blockade of Phe hydroxylation and its conversion to tyrosine. The direct consequences of this impairment are the accumulation of Phe in the body and a decrease in tyrosine formation. At high concentrations of Phe, alternative metabolic pathways are activated, producing phenylpyruvate, phenylacetate, phenyllactate, and other derivatives that exert toxic effects on various organs and tissues [16, 17].

Central nervous system (CNS) structures are affected to the greatest extent. Brain damage is associated with the effects of excess Phe: an imbalance of amino acids in brain tissues necessary for protein synthesis, caused by the inhibition of their absorption in the gastrointestinal tract or impaired reabsorption from renal tubules under conditions of excess Phe in body fluids, impaired formation or stabilization of polyribosomes, and reduced synthesis of myelin, norepinephrine, and serotonin, which play an exceptionally important role in the maturation and functioning of the CNS [14, 16, 17].

Phe is a competitive inhibitor of tyrosinase, the key enzyme in melanin synthesis. The blockade of this pathway, along with reduced availability of tyrosine (the precursor of melanin), leads to hypopigmentation of the hair and skin [3].

The activity of the PAH enzyme depends on three main cofactors: PAH-stimulating protein (PAHSP), tetrahydrobiopterin (BH4), and molecular oxygen. The function of BH4 is to stabilize the quaternary structure (folding) of PAH and other enzymes involved in the hydroxylation of tyrosine, tryptophan, and arginine (Fig. 2).

Fig. 2. The role of tetrahydrobiopterin in the hydroxylation of aromatic amino acids.

1.3 Epidemiology of the Disease or Condition

The prevalence of HFA among the world's population varies significantly depending on the population: from 1:4370 in Turkey to 1:80500 in Japan. The highest prevalence of the disease is noted among the Caucasian race [3–5]. According to European HFA screening centers, the prevalence of the disease in the Eastern European population is higher than in Western and Southwestern European populations. In Ireland, the prevalence of HFA is 1:4500–1:7300, in Italy 1:12280, and in Greece 1:18640 [5]. In Scandinavian populations, the prevalence of HFA is exceptionally low, especially in Finland (1:71000) and Sweden (1:43230) [4, 5].

In Russia, according to neonatal screening data, the prevalence of HFA is 1:7000 and ranges across various regions from 1:4735 in Kursk Oblast to 1:18000 in the Republic of Tyva. In Saint Petersburg, the HFA prevalence is 1:7600, and in Moscow 1:5600. PAH-deficient HFA is detected in 97–98% of cases. BH₄-deficient HFA accounts for 1–3% of cases detected during neonatal screening [8].

1.4 Features of Coding the Disease or Condition

E70.0 Classical phenylketonuria

E70.1 Other hyperphenylalaninemias

E70.9 Disorders of aromatic amino acid metabolism, unspecified

Comments: Classical phenylketonuria (E70.0) includes HFA caused by pathogenic variants in the PAH gene. Other hyperphenylalaninemias (E70.1) include BH₄-deficient HFA.

1.5 Classification of the disease or condition

There are several approaches to the classification of HFA. PAH-dependent and PAH-independent forms of HFA are distinguished. Depending on the severity and maximum blood Phe concentration before treatment after establishing a clinical diagnosis or newborn screening, mild, moderate, or severe forms of HFA are identified (Appendix G1).

PAH-dependent HFA (phenylketonuria) is caused by a defect in the phenylalanine hydroxylase (PAH) enzyme, which converts Phe to tyrosine with the participation of the cofactor/chaperone BH₄. PAH dysfunction results from mutations in the PAH gene located in the 12q22-q24.2 region. This nosology is the most common form, occurring in 98% of all HFA cases [6, 7].

Depending on severity, the following forms are distinguished:

The mild form of HFA requires monitoring and differential diagnosis. Strict dietary treatment for this form of HFA is generally not prescribed. Patients with the mild form of HFA must remain under systematic medical observation with monitoring of blood Phe levels [3, 16, 42].

The moderate form of HFA implies the preservation of partial PAH enzyme activity, requires adherence to a low-phenylalanine diet, and a responsiveness test to the synthetic tetrahydrobiopterin analogue sapropterin** in order to prescribe it in combination with a low-phenylalanine diet [16, 28].

The severe form of HFA is caused by minimal PAH enzyme activity. It requires strict adherence to a low-phenylalanine diet, and in some cases (after genotype determination), a responsiveness test to sapropterin** (ATC code A16AX07) to identify the patient's potential sensitivity to cofactor therapy.

Based on molecular genetic research data, a modern classification of HFA has been created that reflects the etiopathogenesis of the disease (Appendix A3.2) [6-13].

PAH-independent HFA includes several genetically heterogeneous forms (obsolete term «atypical PKU»):

1. BH₄-deficient HFA (type A) is caused by 6-pyruvoyltetrahydropterin synthase (PTPS) deficiency, which is involved in the synthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. The disease is caused by a mutation in the structural gene PTS of cytosolic 6-pyruvoyltetrahydropterin synthase, accompanied by enzymatic deficiency of PTPS in the liver and erythrocytes. The PTS gene is located on the long arm of chromosome 11, in region q22.3-23.3 [6, 7, 9, 34, 35].

2. BH₄-deficient HPA (type B) is caused by GTP cyclohydrolase 1 (GTPCH) deficiency, which functions at the initial stages of BH₄ synthesis by converting guanosine 5'-triphosphate into dihydroneopterin triphosphate. The enzyme is controlled by the GCH1 gene located on the long arm of chromosome 14, in region 14q22.2 [6, 7, 9, 34, 35].

3. BH₄-deficient HPA (type C) is caused by dihydropteridine reductase (DHPR) deficiency, which controls the regeneration of BH₄ from dihydrobiopterin, ensuring its reactivation. The disease is caused by a mutation in the QDPR gene, which is located on the short arm of chromosome 4, in region 4p15.3 [6, 7, 9, 34, 35].

4. BH₄-deficient HPA (type D) is caused by pterin-4-alpha-carbinolamine dehydratase (PCD) deficiency, which is also involved in BH₄ regeneration. The enzyme is encoded by the PCBD1 gene located on the long arm of chromosome 10, in region 10q22.1 [6, 7, 9, 34, 35].

5. DOPA-responsive dystonia, caused by sepiapterin reductase (SPR) deficiency, is conventionally included in the HPA group, as this condition does not typically present with elevated blood Phe levels. Nevertheless, Phe levels are elevated in the patient's brain, which leads to the development of a clinical picture similar to other BH₄-deficient HPAs. The disease is associated with a deficiency of the SPR enzyme, which takes part in the final stage of BH₄ synthesis. Sepiapterin reductase activity is encoded by the SPR gene located on the long arm of chromosome 2, in region 2q13.2 [6-8].

6. Hyperphenylalaninemia without tetrahydrobiopterin deficiency is caused by mutations in the DNAJC12 gene, which encodes the JDP1 protein. The JDP1 protein performs a function similar to BH₄—it acts as a chaperone for enzymes involved in neurotransmitter metabolism and PAH enzyme folding. Thus, a defect in the DNAJC12 gene leads to the development of a phenotype similar to BH₄-deficient HPAs [6-8].

1.6 Clinical presentation of the disease or condition

Clinical features of PAH-deficient HPA (classical PKU).

The clinical manifestations of the disease depend on the timing of therapy initiation and the severity of enzyme impairment. Without effective therapy, the majority of patients with significant PAH deficiency, known as classical PKU, develop severe nervous system damage, whereas timely initiation of therapy makes it possible to avoid pronounced clinical manifestations of the disease [21].

Patients with severe HPA (classical PKU) without therapy.

In the first weeks of life, infants with HPA appear healthy. Manifestation of the disease occurs at the age of 2–6 months in the absence of dietary therapy, characterized by lethargy, absence/loss of interest in the surroundings, or conversely, increased irritability, restlessness, muscle hypertonia, hyperreflexia, and eczematous rash. A characteristic "mousy" odor of sweat and urine is observed, which is explained by the excretion of phenylacetate and phenylpyruvate. In the second half of life, a regression in motor and psychocommunicative development is observed: children stop responding to addressed speech, recognizing their mother, fixing their gaze, and responding to bright toys, and fail to roll over onto their abdomen or sit up. Physical development is impaired to a lesser extent, but a slight decrease in head circumference or even microcephaly, and delayed tooth eruption are detected. Most untreated children have fair hair and blue eyes. The skin is almost entirely devoid of melanin and has increased sensitivity to insolation and trauma; therefore, severe eczema, dermatitis, follicular keratosis, and an increased susceptibility to pustular infections are frequently observed.

In the absence of treatment, severe intellectual disability develops (IQ<50). Epileptic seizures of the "salaam" attack, "nodding", and absence types occur in 50% of patients and are resistant to anticonvulsant therapy. Epileptic activity during electroencephalography is recorded in 85–90% of patients. Other neurological disorders are also possible—ataxia, hyperkinesia, hand tremor, central paresis, which are associated with impaired neuronal myelination processes and decreased white matter density in the brain. Due to chronic metabolic disorders, the production of neurotransmitters (dopamine, serotonin, norepinephrine) is reduced.

Currently, clinical manifestations of HPA are practically non-existent due to early diagnosis of the disease and timely initiation of treatment. However, in rare cases, patients may be "missed" during mass screening or parents may fail to adhere to the prescribed diet; therefore, HPA should be ruled out in unclear cases of intellectual disability in children.

Patients with severe PAH-deficient HPA undergoing treatment.

Despite early initiation of treatment and adherence to the diet, patients may experience cognitive impairments characterized by peculiarities in visual and executive functions, reduced information processing speed, low attention span, and impairments in the dynamics of memorization and speech. Even with a normal level of intellectual development, various emotional alterations that hinder their full social adaptation are identified in 96% of children with HPA: reduced cognitive abilities, high levels of anxiety, emotional instability, hyperactivity, and aggressiveness [57, 59, 66].

Among patients who received a low-phenylalanine diet with the inclusion of unbalanced medical nutrition products, there are 4 times more children with overweight and obesity, and 12 times more with malnutrition compared to patients treated with modern specialized amino acid-based products free of Phe. With late initiation of dietary therapy, in addition to persisting neurological symptoms, an increase is noted in the number of children with short stature (10-fold), malnutrition (6.5-fold), and obesity (7-fold) compared to the group of patients in whom treatment was initiated in a timely manner [67].

Against the background of elimination dietary therapy, deficiencies of trace elements (zinc, selenium, iron, copper, and magnesium), carnitine, ω-3 polyunsaturated fatty acids, and vitamins B6, B12, and B9 (folic acid) may occur [61-63]. Numerous studies have proven a high frequency of osteopenia in patients with HPA compared to the general population. However, the exact mechanisms of bone mineral density reduction have not been established. A relationship has been revealed between bone resorption/formation processes and adherence to dietary therapy and adequate intake of trace elements from special nutritional products [68].

Assessment of the quality of life of children with HPA has shown that all its parameters—physical, emotional, and social functioning, and functioning at school/kindergarten—are lower compared to healthy peers. Maximum differences are established in schoolchildren, which, according to the authors, is associated with an increase in mental and physical load, and a rise in internal psycho-emotional tension. At the same time, it has been shown that the later treatment is initiated in HPA patients, the lower the indices of social and role functioning, and the older the examined children, the lower the indices of their adaptation in the peer group [69].

The correlation between elevated Phe levels in early life and long-term reduction in IQ is well studied. The impact of Phe levels on IQ throughout life has also been demonstrated in studies on adults. Thus, in those who reduce their adherence to a Phe-restricted diet in adulthood, IQ drops slightly, further supporting the concept of a "lifelong" diet [4, 57].

Nevertheless, even with strict adherence to a low-Phe diet in adulthood, cognitive impairments may persist, such as decreased language skills, memory, perception and information processing speed, learning ability, and executive function [58].

Adults who do not follow a Phe-restricted diet and have elevated blood Phe levels typically have poor concentration and demonstrate slowed motor reactions [29, 58].

There is also a higher level of anxiety, depression, phobias, and panic attacks in individuals treated in childhood but who discontinued therapy in the second decade of life. These findings appear to be related to both current and anamnestic blood Phe levels [3, 36, 60, 66, 67].

Patients with a mild biochemical and clinical phenotype of PAH-deficient HPA.

It is believed that in PAH-deficient HPA, even if patients without dietary therapy have plasma Phe concentrations consistently below 600 µmol/L (10 mg/dL; units of Phe measurement: 1 mg/dL corresponds to 1 mg%, 60 µmol/L), the risk of developing intellectual, neurological, and neuropsychological impairments is higher than in individuals without PAH deficiency. Experts recommend Phe restriction for any patient with a Phe level > 360 µmol/L (6 mg/dL) [5, 17].

Cases of HPA with mild PAH deficiency have been described in patients with normal intelligence whose diagnosis was established in adulthood during a sudden deterioration of their mental state [41, 60].

"Maternal phenylketonuria".

Maternal PKU syndrome is a set of symptoms occurring in children born to women with hyperphenylalaninemia (HPA) who did not receive (or only partially received) specialized treatment (diet, etc.) during the preconceptional period and pregnancy. The syndrome develops as a result of the fetus being exposed to elevated maternal blood phenylalanine (Phe) levels (above 360 µmol/L) and its metabolites. The following manifestations of maternal PKU syndrome are distinguished [4]:

- intrauterine growth restriction (IUGR). The incidence does not differ from the general population if maternal Phe levels are controlled during the first ten weeks of pregnancy and do not exceed the recommended level of 360 µmol/L; the risk of fetal IUGR increases if Phe concentrations are optimized later;

- microcephaly. The risk is 5-18% during pregnancies where the mother's Phe level is optimized before pregnancy and during the first ten weeks. The risk increases to 67% if appropriate Phe levels are not achieved within 30 weeks of pregnancy;

- congenital heart disease and other malformations. Elevated maternal blood Phe concentrations (> 600 µmol/L) during early pregnancy lead to an approximately 8%-12% risk of cardiac malformations. Phenotypic features and other congenital defects (including tracheoesophageal fistulas, etc.) have also been reported in infants born to women with HPA;

- intellectual disability (>90%). The critical threshold is a maternal blood Phe concentration consistently exceeding 360 µmol/L during pregnancy, with an inverse relationship between the child's cognitive function and maternal Phe levels above 360 µmol/L;

With ongoing treatment and monitoring of blood Phe levels, the risk of congenital fetal malformations in a woman with HPA is minimal.

Clinical features of BH₄-deficient HPA

Clinically, BH₄-deficient HPA are characterized by hyperphenylalaninemia, deficiency of the neurotransmitters dopamine and serotonin, and progressive intellectual and motor disorders [4, 6]. These nosological forms are characterized by a lack of positive clinical response to dietary Phe restriction despite an adequate decrease in blood Phe levels. Without treatment, BH₄-deficient HPA types A, B, and C share similar clinical symptoms and are characterized by early manifestation within the first 3 months of life, and in half of the cases, in the neonatal period. The severity of clinical manifestations in children and adults varies. In severe cases, the clinical picture is dominated by rapidly progressive psychomotor retardation, tonic-clonic seizure attacks, hyperexcitability, hyperreflexia, muscular dystonia (truncal muscle hypotonia, limb muscle hypertonia), spastic tetraparesis, as well as extrapyramidal symptoms (choreiform hyperkinesia), gait disturbance, tremor, ataxia, swallowing disorders, hypersalivation, thermoregulation disorders, pseudobulbar disorders, and oculogyric crises (episodic conjugate eye deviation, usually directed upward and laterally). In some cases, neurological symptoms fluctuate throughout the day: worsening in the afternoon and improvement in the morning after sleep. Microcephaly is common in types A and C BH₄-deficient HPA. Mild forms of BH₄-deficient HPA may present with minimal clinical signs.

Elevated plasma Phe concentrations in patients with BH₄-deficient HPA can vary widely from mild (no more than 360-600 µmol/L) to very high (2500 µmol/L), similar to classic PKU.

In some patients with GCH1 gene defects, blood Phe levels at birth may be normal (such cases will not be detected by newborn screening), but Phe concentrations may periodically rise later. A sharp decrease in the levels of the end metabolites of biogenic amines—homovanillic and 5-hydroxyindoleacetic acids—is detected in the tissues and biological fluids of the affected patients.

6-pyruvoyl-tetrahydropterin synthase deficiency (BH₄-deficient HPA type A).

This variant is the most common, accounting for about 60% of all BH₄-deficient HPA cases. It manifests in severe (central) and moderate (peripheral) forms. The former, most common (80%) severe form is accompanied by a marked decrease in the levels of the biogenic amines serotonin and catecholamines in the cerebrospinal fluid (CSF).

In the severe form, nonspecific symptoms may appear from the first weeks of a child's life: muscle hypotonia, weak sucking, and decreased motor activity. Some infants are born with signs of intrauterine growth restriction. Central nervous system disorders typically become noticeable at about 4 months of age. Without pathogenetic therapy, patients experience drowsiness, irritability, muscular dystonia, hyperkinesia, hyperthermia, hypersalivation, and swallowing difficulties, followed later by seizures (tonic-clonic, myoclonic) and intellectual disability. Microcephaly is frequently observed in this form [70]. The peripheral form is characterized by a mild decrease in neurotransmitter levels, mild HPA, a more favorable clinical course with late onset, and moderate CNS involvement. Deterioration of the patient's condition usually occurs during adolescence and manifests as neurovegetative and pain attacks.

GTP cyclohydrolase I deficiency (BH₄-deficient HPA type B), which leads to the clinical presentation of BH₄-deficient HPA B [6], also causes DOPA-responsive dystonia, which can be inherited in an autosomal dominant manner. Patients exhibit progressive neurological symptoms that are usually milder than those of untreated classic PKU.

Dihydropteridine reductase deficiency (BH₄-deficient HPA type C). The clinical manifestations of this form are similar to those of other forms of BH₄-deficient HPA.

Pterin-4-alpha-carbinolamine dehydratase deficiency (BH₄-deficient HPA type D) is characterized by a benign, mild clinical course [6].

Sepiapterin reductase deficiency (DOPA-responsive dystonia). Reported cases of SPR gene defects are not accompanied by hyperphenylalaninemia. Clinical manifestations are similar to BH₄-deficient HPA [64].

Nonspecific symptoms may be present in infancy: motor development delay and low muscle tone. The disease usually develops in the first year of life, presenting with dystonia, delayed development of motor and speech skills, muscle weakness, central hypotonia, and oculogyric crises; symptom severity fluctuates throughout the day (worsening at night, improvement in the morning after sleep). Sleep disturbances and psychological disorders (anxiety, irritability) frequently develop later. Intellectual impairment often occurs, but many patients experience only mild or moderate learning difficulties. Symptoms such as tremor, bradykinesia, muscle rigidity, masked facies, dysarthria, hyperreflexia, increased limb muscle tone, and autonomic dysfunction develop less frequently. In many cases, dystonia and pronounced symptom fluctuations throughout the day appear only at a later stage of the disease.

Since this form of the disease does not involve elevated blood Phe, and therapeutic approaches differ fundamentally from all other forms of Phe metabolism disorders, a more detailed description of sepiapterin reductase deficiency is not provided in these clinical guidelines.

HPA without tetrahydrobiopterin deficiency caused by mutations in the DNAJC12 gene.

This form is typically detected during newborn screening. Clinical symptoms are similar to BH₄-deficient forms: psychomotor retardation, hyperexcitability, hyperreflexia, muscular dystonia, and varying degrees of intellectual disability at an older age. Some patients develop a DOPA-responsive parkinsonian syndrome in childhood. Laboratory testing reveals dopamine and serotonin deficiency in the cerebrospinal fluid and normal tetrahydrobiopterin metabolism [65].

Thus, six forms of HPA can be distinguished, differing in approaches to diagnosis, treatment, and monitoring: PAH-deficient HPA, mild form (not requiring therapy up to one year of age); PAH-deficient HPA, moderate form requiring therapy; PAH-deficient HPA, severe form requiring therapy; PAH-deficient HPA responsive to sapropterin therapy**; BH₄-deficient HPA; maternal PKU (classic phenylketonuria, pregnancy).

2. Diagnostics, medical indications and contraindications for diagnostic methods

Criteria for diagnosis.

The main diagnostic criterion for HPA is an elevated blood phenylalanine level above 2.0 mg/dL (120 µmol/L), which is primarily detected during neonatal screening.

The diagnosis of HPA is confirmed in the presence or absence of characteristic clinical manifestations of the disease combined with an elevated blood phenylalanine level. To verify the diagnosis, additional molecular genetic testing and determination of the phenylalanine/tyrosine ratio are required.

Please note that, in accordance with the requirements for the development of clinical guidelines, each recommendation statement must indicate the strength of the recommendations and the evidence base in accordance with the scales for assessing the level of reliability of evidence (LRE) and the level of conviction of recommendations (LCR). For many statements, the LCR and LRE will be low due to the lack of high-design clinical studies dedicated to them. Despite this, they are necessary elements of patient examination to establish a diagnosis and choose treatment tactics.

2.1 Complaints and Anamnesis

Most patients with HPA are diagnosed during mass newborn screening, and with the timely prescription of specialized dietotherapy, the disease proceeds asymptomatically.

The spectrum of possible complaints in patients with HPA who were diagnosed late or in the absence of treatment can be quite wide and depends on the form of the disease (classic or non-classic) and individual features of the disease course [6].

Complaints in PAH-deficient HPA (classic PKU): global developmental delay, growth retardation, speech developmental delay, social developmental delay, intellectual disability, behavioral abnormalities, attention deficit hyperactivity disorder, autoaggression, depression, autism, memory impairment, seizure attacks, muscular hypotonia, limb hypertonia, tremor, loss of motor skills, nausea, vomiting, abnormal urine odor, loss of skin elasticity, skin and hair hypopigmentation, eczema, cataract, microcephaly, hyperphenylalaninemia and decreased PAH activity according to laboratory tests, brain calcifications, white matter brain lesions based on MRI results.

Complaints in BH₄-deficient HPA (may persist despite adherence to a Phe-restricted diet): breastfeeding difficulties, motor developmental delay, microcephaly, hypersalivation, swallowing disorders, drowsiness, ptosis, febrile episodes, extrapyramidal disorders (parkinsonism, choreoathetosis, chorea), episodes of brief rapid muscle contraction.

Complaints and anamnesis are also described in the "Clinical presentation" section.

2.2 Physical Examination

Comment: it is necessary to assess somatic and neurological status, and basic anthropometric parameters.

2.3 Laboratory Diagnostics

Comments: the neonatal screening scheme is given in Appendix B. Screening examination for HPA meets all accepted criteria for mass screening, including cost-benefit ratio. Dried blood spots are sent for screening examination within the established timeframes to a specialized neonatal screening laboratory of a specific region. Commonly accepted reference values for blood Phe are 0-120 µmol/L (0-2 mg/dL).

Comment: in HPA, the Phe concentration exceeds 120 µmol/L (2 mg/dL). Normally, the phenylalanine/tyrosine ratio is less than 1; a ratio greater than 3 indicates a high probability of PAH deficiency [4]. More severe forms of PAH deficiency with complete loss of enzyme activity (often referred to as "classic PKU") are accompanied by an increase in Phe concentration of more than 1200 µmol/L (20 mg/dL) [5].

Comment: molecular genetic testing of the PTS, QDPR, GCH1, PCBD, SPR, and DNAJC12 genes is also performed to clarify the diagnosis and conduct differential diagnosis. The set of tests for the diagnosis of phenylketonuria is included in the nomenclature of medical services.

The algorithm for DNA diagnostics of HPA is presented in Appendix A3.4.

Molecular genetic diagnostics (DNA diagnostics) can be performed using the following methods:

1. Screening for common PAH gene mutations [8];

2. Sanger sequencing of the PAH, PTS, QDPR, GCH1, PCBD, SPR, and DNAJC12 genes;

3. Panel, whole-exome, or whole-genome next-generation sequencing followed by confirmation of results by Sanger sequencing [9, 10].

Genotyping results are interpreted by a medical geneticist and used for:

1. Family counseling, determining the risks of hereditary pathology for the proband's relatives, and pregnancy planning;

2. Differential diagnosis of PKU, BH₄-deficient HPA types A, B, C, D, DOPA-responsive dystonia, and non-BH₄-deficient HPA [8, 9, 10];

3. Determining the potential sensitivity or potential resistance of patients with PKU to therapy with pharmacological tetrahydrobiopterin analogues [11, 12, 13].

Based on genotype data, one can judge the patient's potential response to therapy with pharmacological tetrahydrobiopterin analogues (sapropterin**, BH₄). Provided that the pathogenic variant of at least one of the alleles leads to the formation of an enzyme protein with residual activity, the patient may be recommended to undergo a loading test with BH₄ (sapropterin**).

Comment: diagnostic reference values for phenylalanine and pterins in blood, urine, and cerebrospinal fluid are presented in Appendix A3.3 [6, 7]. Since BH₄ deficiency encompasses a group of disorders that can be detected by neonatal HPA screening but cannot be identified by routine screening methods, selective screening for BH₄ deficiency is necessary for every newborn with a plasma phenylalanine level above 120 µmol/L, as well as for older children with HPA and neurological symptoms who do not have pathogenic variants in the PAH gene.

Comments: PAH enzyme activity can only be determined in liver and kidney tissue obtained via biopsy. Due to the high risk of complications, this method is not used for screening or other diagnostic tests [4].

Comments: a complete blood count (CBC) includes measuring blood levels of total hemoglobin, erythrocytes, leukocytes, and platelets, evaluating hematocrit and the erythrocyte sedimentation rate (ESR), and performing a differential leukocyte count (leukocyte formula).

Comments: a routine general biochemical blood test includes determining the blood activity of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, and gamma-glutamyl transferase; measuring blood levels of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total bilirubin, cholesterol, triglycerides, albumin, glucose, creatinine, urea, total protein, sodium, potassium, total calcium, ionized calcium, inorganic phosphorus, and ferritin; and determining protein fractions by electrophoresis.

Comments: restrictive diets may lead to essential amino acid deficiency, particularly tyrosine. The test is performed using tandem mass spectrometry.

2.4 Instrumental Diagnostics

Comment: brain MRI may be performed under combined inhalation anesthesia. Progressive white matter lesions are observed in 90% of patients with HPA even in the absence of neurological disorders. With age, untreated patients or those who discontinued the diet show more pronounced MRI changes. It is believed that high Phe levels in untreated patients impair neuronal myelination, whereas early treatment preserves normal myelin formation. Damage to the myelin sheath can occur due to elevated Phe levels during diet relaxation or discontinuation, as well as due to poor patient compliance [14]. The frequency of the examination is determined by the specialist physician depending on the clinical status.

Comment: brain CT may be performed under combined inhalation anesthesia. In the absence of treatment, calcifications form in the basal ganglia of the brain and can be detected by computed tomography. The study is usually performed in children older than 3 years. In HPA patients with an open fontanelle, neurosonography may be performed to detect brain pathology. The frequency of the examination is determined by the specialist physician depending on the clinical status.

Comments: the frequency of the examination is determined by the specialist physician depending on the clinical status.

Comments: renal ultrasound (US) is usually performed as a screening and dynamic follow-up method in patients with HPA to detect dysmetabolic nephropathies and nephrocalcinosis. The frequency of the examination is determined by the specialist physician depending on the clinical status.

Comments: the frequency of the examination is determined by the specialist physician depending on the clinical status.

Comment: Electrocardiography is performed in patients with all forms of HPA, and echocardiography is performed as clinically indicated. The frequency of examinations is determined by the specialist physician based on the patient's clinical status.

Comments: the frequency of the examination is determined by the specialist physician depending on the clinical status.

Comments: expert-level fetal ultrasound is indicated. All pregnant women with HPA undergo screening ultrasound at 11–14 weeks, 18–21 weeks, and 30–34 weeks of gestation, as well as additionally at 15–16 weeks and 25–26 weeks of gestation. The frequency of the examination is determined by the specialist physician based on the clinical status.

2.5 Other Procedures

Specialist consultations may be provided to patients at various stages of medical care, including during the disease diagnosis period.

Comments: medical history and complaints are collected; pedigree analysis is performed; visual examination is conducted along with anthropometry: measurement of height, weight, and head circumference. Frequency depends on the patient's age. The initial patient evaluation should be performed no later than 3 weeks of the child's life. The optimal monitoring frequency, according to European guidelines, is:

- under 1 year of age — at least once every 2 months

- from 1 year to 12 years — at least once every 6 months

- from 12 to 18 years — at least once every 6 months

- over 18 years — at least once a year

- during pregnancy — at least once per trimester [16].

According to the Russian expert working group, it is advisable to conduct more frequent monitoring in patients under 1 year of age — at least once a month; from 1 year — at least once every 3 months; from 7 years — at least once every 6 months; over 18 years — at least once a year; in pregnant women — depending on the gestational age, at least once every 2 weeks.

If this cannot be accomplished due to the remote place of residence of the child, telecommunication contact with the patient must be established [16].

To improve compliance, the legal representatives of a child with HPA may complete an informed consent form regarding the necessity of following the doctor's requirements and treatment (Appendix A3.5).

Comments: anamnesis and complaints are collected; visual examination with anthropometry: measurement of height, weight, and head circumference.

Comments: consultation with a clinical nutritionist is indicated for patients to provide specialized medical care. If the staff schedule does not include a clinical nutritionist, dietary counseling is provided by a medical geneticist or gastroenterologist. Consultation is required during the initial examination and subsequently at least once a month for 12 months.

Comments: a cardiological evaluation is necessary, including echocardiography (at initial assessment and subsequently as clinically indicated) and electrocardiogram recording, with follow-up as indicated.

Comments: patients require testing using approved methods to assess intellectual development. It is advisable to perform this starting from 6 months of age and subsequently at least once every 6 months, and in adults — at least once a year.

3. Treatment, including drug and non-drug therapies, diet therapy, and pain management

The main goal of treatment is normal neurocognitive and physical development, as well as psychosocial adaptation of the patient. The best way to achieve this goal is to reduce and regularly monitor blood phenylalanine levels in patients with HPA, striving to keep this indicator within the target therapeutic range. The management of patients with HPA involves a multidisciplinary approach with the mandatory participation of geneticists, dieticians, general practitioners (therapists), pediatricians, neurologists, cardiologists/pediatric cardiologists, gastroenterologists, medical psychologists, psychiatrists, physical therapists, endocrinologists/pediatric endocrinologists, and specialists in other fields experienced in treating this rare disease [16].

Comments: most specialists believe that treatment in newborns should be initiated before the age of 10 days [16]. Considering that a delay in treatment initiation of every 4 weeks led to a decrease in the IQ score by approximately 4 points [18], it is advisable to start treatment no later than 3 weeks of life.

Comments: treatment should not be prescribed to previously untreated patients with a blood phenylalanine concentration <360 µmol/L. Regular monitoring (at least once a month) of blood phenylalanine levels must be performed in these patients in order to timely prescribe treatment if phenylalanine levels rise [16].

Comments: studies have shown that maintaining blood phenylalanine concentrations below 360 µmol/L in patients from birth to 12 years and 400–600 µmol/L in patients over 12 years and adults preserves satisfactory neurocognitive development, adequate information processing speed, and psychosocial functions. Therefore, lifelong treatment is recommended, including lifelong adherence to dietary restrictions with the use of specialized protein substitutes, vitamin and mineral sources [19].

3.1 Conservative Treatment

3.1.1 Diet Therapy

Comments: diet therapy is based on a drastic restriction of phenylalanine in the patient's diet through the exclusion of high-protein foods and their replacement with specialized therapeutic products that are partially or completely free of phenylalanine. Natural foods with a high protein (and consequently phenylalanine) content are excluded from the diet of a hyperphenylalaninemia patient: meat, meat products, fish, fish products, curd, eggs, legumes, nuts, chocolate, etc. Natural foods allowed in the diet are introduced in accordance with the calculated phenylalanine content.

For infants (from birth to 12 months), permitted foods include breast milk, infant formulas, vegetables, fruits, and certain low-protein complementary foods. Complementary feeding is introduced at the same time as for healthy infants (from 4 to 6 months), but using only low-protein foods, taking into account the patient's tolerance to natural protein/phenylalanine [21]. To compensate for the dietary protein component, specialized phenylalanine-free amino acid formulas are used, balanced in composition similarly to adapted infant formulas (see Appendix A3.6).

In the diet of patients older than 1 year, specialized phenylalanine-free amino acid formulas with a high protein content, fortified with vitamin and mineral complexes, are generally used. Formulas intended for children aged 1 to 8 years may contain sufficient amounts of carbohydrates and fats. This allows for the individualized selection of the therapeutic product depending on the nutritional status of the patients, the functional state of the gastrointestinal tract, and food preferences. Furthermore, these amino acid formulas, including those for infants, being more balanced in composition, can also be used in older patients and pregnant women with hyperphenylalaninemia in conditions caused by gastrointestinal diseases and intolerance to certain foods.

Comments: low-protein foods are indicated for children over 1 year of age and subsequently for all patients with hyperphenylalaninemia. The list of low-protein and protein-free specialized products registered in the Russian Federation is provided in Appendix A3.7. It is recommended when initiating diet therapy to titrate the amount of dietary phenylalanine (natural protein) for all patients with hyperphenylalaninemia under the control of blood phenylalanine to achieve target therapeutic values [22].

GCR CLEL 5Grade of Recommendation C (Level of Evidence – 5).

Comments: a reduction in the amount of daily protein (but not more than 10%) is allowed, mainly at the expense of the amino acid formula protein, depending on tolerance to food and phenylalanine, as well as the patient's physical activity and nutritional status [19, 24].

Protein from natural foods in the diet is calculated based on the permissible daily amounts of phenylalanine, taking into account that 1 g of protein contains ~50 mg of phenylalanine. Depending on dietary phenylalanine tolerance, its permissible and safe daily amount ranges from 90 to 35 mg/kg of body weight for infants. In the diet of children over 1 year of age, as well as adolescents and adults, the permissible amount of phenylalanine gradually decreases from 35 to 10 mg/kg of the child's body weight (Appendix A3.9, Appendix A3.10). Also, during treatment, daily tolerance to dietary phenylalanine is determined, which depends on the degree of impairment of the phenylalanine hydroxylase enzyme and the patient's age.

Comments: when prescribing a diet, an individual and differentiated approach to the use of specialized and natural products according to the patient's age is important.

For patients with hyperphenylalaninemia, regardless of their age, the restriction on foods richest in phenylalanine, such as meat, fish, and products thereof, remains in effect. Curd, hard cheeses, legumes, chicken eggs, and nuts can be included in limited quantities in the diet of older patients, taking into account phenylalanine tolerance. Consumption of fast food and carbonated beverages with sweeteners (aspartame or food additive E951) containing phenylalanine must be excluded for patients with hyperphenylalaninemia.

The tactics of diet therapy for concomitant diseases (severe hyperthermia, intoxication, various dyspeptic symptoms), as well as in case of refusal to take the amino acid formula, consist of a short-term (2–3 days) cessation of diet therapy, replacing therapeutic products with natural ones with a low protein content. After the acute period of the disease subsides, the specialized product is reintroduced into the diet, but over a shorter period than at the beginning of treatment. If a patient with hyperphenylalaninemia does not refuse food during illness, the treatment of concomitant somatic diseases is carried out according to the standard scheme and does not require stopping diet therapy.

3.1.2 Drug therapy

Comments: depending on the form of hyperphenylalaninemia, sapropterin** is used in combination with diet therapy or without it (Appendix A3.11). The starting dose of sapropterin** in patients with BH₄ deficiency ranges from 2 to 5 mg/kg of body weight taken once daily. The dose can be increased up to 20 mg/kg of body weight per day. To achieve the optimal therapeutic effect, the daily dose of the drug can be divided into 2 or 3 doses throughout the day.

Comments: levodopa+[carbidopa]** tablets are prescribed for the indication "parkinsonism syndrome" (contraindicated in children under 18 years of age, dosage is selected individually, usually 10−15 mg/kg per day calculated by levodopa is required)

Comment: folic acid** is prescribed to patients with a deficiency of this micronutrient in accordance with the drug's instructions for use (it is especially important to prescribe folic acid** in the first trimester of pregnancy – 400 mcg/day).

In some cases, a diet with restriction of phenylalanine (PA) and the administration of folic acid and its derivatives is required (Appendix A3.11).

Administration should be carried out under the control of blood folic acid levels; a number of studies even note elevated folic acid levels in patients with phenylketonuria (PKU) [121].

Comments: testing for potential drug sensitivity and treatment with sapropterin** are performed and monitored by the physician who also provides follow-up care for patients with HPA (testing regimens: Appendix A3.12).

Response to treatment with sapropterin** is assessed by the degree of decrease in the patient's blood PA concentration while maintaining a stable low-phenylalanine diet. A patient is considered responsive if the difference between the PA level obtained at the end of the treatment response assessment period and the baseline level before starting the drug is 30% or more.

Comments: if a positive response is obtained in the form of a decrease in blood PA concentration and an increased tolerance to dietary phenylalanine, a trial treatment of at least 28 days or more is carried out, during which the degree of increase in dietary phenylalanine tolerance is determined and further treatment tactics are developed. Testing may be performed in newborn patients.

Comments: some patients exhibit a delayed positive response to sapropterin** treatment (so-called "late responders"). If necessary, long-term pharmacotherapy in patients with HPA who respond to sapropterin** treatment by lowering blood PA levels is carried out in combination with a diet using amino acid mixtures, the amount of which is determined by the physician.

With the patient's consent, continuation of sapropterin** therapy during pregnancy is possible: sapropterin** is prescribed to women with HPA who have previously demonstrated sensitivity to sapropterin** treatment or those who have previously received treatment with this drug.

Comments: cholecalciferol** is usually prescribed in accordance with guidelines for the correction of vitamin D deficiency. Prescription is carried out after consultation with an endocrinologist and under the control of blood vitamin D levels.

3.2 Surgical Treatment

Surgery is usually not required.

3.3 Other Treatment

3.3.1 Training

Education is an integral part of the complex of therapeutic measures for HPA and should continue throughout the entire course of the disease. HPA treatment necessarily includes training in self-monitoring of PA levels and principles of disease management (calculation of diet therapy).

Comments: the goals and objectives of education must be specified in accordance with the patient's current condition. Patients who have not undergone training (primary cycle) or patients who have already completed training (repeat cycles) are referred to phenylketonuria school to maintain their level of knowledge and motivation or when new therapeutic goals emerge.

Comments: training can be conducted either individually or in patient groups. The content of educational programs must comply with established standards for the diagnosis and treatment of HPA, and their structure must take into account basic pedagogical principles. The programs imply a strictly practical focus and accessibility of perception. Mandatory sections of the educational programs:

1. school for young parents of a child with HPA;

2. general information about HPA;

3. monitoring PA levels;

4. diet therapy;

5. socialization and quality of life;

6. psychological aspects in children and adolescents with HPA;

7. follow-up observation in HPA;

8. preparation for pregnancy and pregnancy in HPA

Most of the time during the training process should be devoted to the practical development of skills necessary for independent disease management. First of all, this concerns monitoring PA levels and the technique of calculating the amount of protein in food.

4. Medical Rehabilitation and Sanatorium-Resort Treatment

The main aspects of medical and social care for patients with HPA are diagnosis, treatment monitoring, and social rehabilitation.

Particularly in school-age children and adults, blood PA levels increase due to non-compliance with dietary recommendations, which requires psychological support for the patient. The patient's level of intellectual development and cognitive functions correlate with blood PA values throughout life [36, 37].

Comments: psychological and pedagogical rehabilitation of adolescents. They face problems of puberty, which is accompanied by a desire for independence, oppositional behavior, and susceptibility to peer pressure. This is a period of frequent refusals by adolescent patients with HPA from diet therapy, consumption of specialized nutritional products, as well as a high risk of losing metabolic control and complete loss of medical supervision.

Social rehabilitation includes organizing meals for children with HPA in general education and wellness institutions; for adults, the opportunity to use specialized medical nutrition products in the workplace.

Public organizations play a special role in supporting patients with hyperphenylalaninemia (HPA). An organization uniting parents of children with HPA and adult patients assists in social rehabilitation, social adaptation, employment assistance, organization of recreation (including in summer camps), leisure, socio-cultural rehabilitation, and other activities, as well as holding parent schools and seminars.

Although HPA is not a life-threatening disease, palliative care is considered when necessary as part of rehabilitation; it includes a system of measures to improve the quality of life of patients with life-threatening and life-limiting diseases, predominantly in the terminal stage of development, and represents an active and holistic approach to care starting from the moment of diagnosis and covering physical, emotional, social, and spiritual aspects until death.

Palliative care for patients with HPA is provided on the basis of relevant regulatory documents (see Appendix A3.1)

5. Prevention and Dispensary Observation

5.1 Prevention

Comments: the carrier frequency of a pathogenic mutation in the PAH gene in the Russian Federation is 1:50. If HPA is detected in a family, relatives should be examined to clarify heterozygous carrier status for mutations in the PAH, PTS, QDPR, GCH1, PCBD, SPR, and DNAJC12 genes.

Comments: prenatal diagnosis is performed in a couple where it has been established that the prospective parents are carriers of a mutation in the same gene: PAH, PTS, QDPR, GCH1, PCBD, SPR, DNAJC12. The probability of having a child with HPA for a woman or man suffering from this disease is realized if the second parent is a heterozygous carrier of the mutant gene (50% risk) or also suffers from HPA (100% risk).

Comments: when planning a pregnancy, it is desirable that the blood Phe level 2-3 months prior to conception and during pregnancy does not exceed 360 µmol/L.

Prevention of fetal pathology, including congenital malformations, in a pregnant woman with HPA

Comments: for details, see the "Diagnosis" and "Treatment" sections.

5.2 Dispensary follow-up of patients with classical phenylketonuria

In order to prevent intellectual, cognitive, speech, and learning impairments, as well as to ensure harmonious physical development, dispensary observation includes the following recommendations:

Monitoring treatment adequacy.

Comments. It has currently been established that blood Phe levels decrease throughout the day, with the highest blood Phe level being reached early in the morning after overnight fasting. Rules for blood sampling on filter paper are provided in Appendix A3.14.

Comments: frequent fluctuations and exceeding the target therapeutic blood Phe values in children under 12 years of age subsequently lead to neuropsychological development disorders and cognitive impairments [29, 16].

Comments: higher blood Phe fluctuations on the diet are permitted, but not exceeding 600 µmol/L [29]. It has been shown that a prolonged elevation of blood Phe levels in adults with BH4-dependent HPA negatively affects the psychoemotional state (mood lability, depression) and leads to impaired attention and other neurocognitive functions [30, 16, 52].

Comments: the absence of dietary restrictions and monitoring of blood Phe levels in women during the pre-pregnancy period and throughout pregnancy significantly increases the risk of developing "maternal PKU syndrome" in the fetus [72-74].

Comments: the widely used lower target blood Phe level of 120 µmol/L is derived from clinical experience describing adverse health consequences in patients with persistently very low blood Phe concentrations [31]. In childhood, these include increased morbidity, low nutritional status, and acrodermatitis enteropathica. In adults, they include malnutrition and psychoemotional disorders; in women of reproductive age, infertility; and in pregnant women with HPA, intrauterine growth restriction and the birth of a small-for-gestational-age infant [25].

Comments: low Phe levels on re-testing require differential diagnosis and exclusion of BH4-deficient forms of HPA [33].

Comments: in BH4-deficient HPA, treatment adequacy is also monitored by blood Phe levels [33, 70].

Comments: these recommendations are based on many years of experience of physicians specializing in the treatment of patients with phenylketonuria in the Russian Federation and were presented in previous clinical guidelines for phenylketonuria. Testing must be carried out with the following frequency: under 3 months of age — once a week (until results are within 120 to 360 µmol/L, and thereafter once every 10 days); from 1 year to 6 years — at least 1–2 times a month; 7 years and older — at least once every 3 months. In European countries, the frequency of recommended testing is higher: under 1 year, once a week; from 1 year to 12 years, once every 2 weeks; over 12 years, once a month; during pregnancy preparation, once a week; during pregnancy, twice a week [16, 66].

Comments: age-specific recommendations for the frequency of blood Phe monitoring must be followed.

Comments: with the advent of new therapeutic options for drug treatment, it is recommended to maintain blood PA levels within 120–360 µmol/L (2–6 mg/dL) for all ages [36-42].

Comments: this includes assessing total hemoglobin, erythrocyte, leukocyte, and platelet counts, hematocrit, erythrocyte sedimentation rate, and differential leukocyte count (leukogram). Restrictive diets may lead to nutritional iron deficiency anemia, which can be latent. The test is performed at least once a year [16, 45].

Comments: restrictive diets may lead to crystalluria. The test is performed at least once a year.

Comments: at least once a year.

Comments: restrictive diets may lead to essential amino acid deficiency, particularly tyrosine. The test is performed using tandem mass spectrometry; the frequency is determined individually, on average at least once a year.

Comments: at least once a year; it is advisable to determine protein fractions by electrophoresis, and test blood levels of glucose, total protein, total bilirubin, cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, creatinine, urea, total and ionized calcium, sodium, potassium, inorganic phosphorus, iron, ferritin, 25-OH vitamin D, and serum folic acid, as well as blood alkaline phosphatase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity, in order to assess the balance of key nutrients during diet therapy, calcium-phosphorus metabolism, and renal and hepatic function with a frequency of once a year; restrictive diets may lead to dysproteinemia, mineral metabolism disorders, and essential micronutrient deficiencies. [44, 45, 46, 47, 49].

Comments: it is necessary to monitor B vitamin levels to prevent deficiency states or hypervitaminosis. The frequency of testing is determined by the attending physician depending on the clinical status. For infants in their first year of life, monitoring every 6 months is possible, followed by at least once a year.

Comments: renal ultrasound is routinely performed as a screening and follow-up tool in patients with HPA at least once a year to detect dysmetabolic nephropathies and nephrocalcinosis.

Comments: comprehensive abdominal ultrasound is routinely performed as a screening and follow-up tool at least once a year in patients with HPA to detect liver and pancreatic abnormalities.

Comments: video-EEG monitoring is also performed when necessary. EEG and EEG monitoring are performed according to indications, and once a year in stable cases.

Comment: brain MRI may be performed under combined inhalation anesthesia. Progressive white matter damage is observed in 90% of patients with HPA, even in the absence of neurological disorders. With age, MRI changes are more pronounced in untreated patients or those who have abandoned their diet. It is believed that high PA levels in untreated patients hinder neuronal myelination, whereas early treatment preserves normal myelin formation. Myelin sheath damage may occur due to elevated PA levels resulting from dietary relaxation or discontinuation, as well as poor patient compliance [14].

Comments: testing frequency is based on clinical indications, but no more than once a year [48, 50].

Comments: the test is performed in patients aged 2 years and older

Comment: electrocardiography is performed in patients with all forms of HPA, on average at least once a year, with echocardiography performed as clinically indicated.

Comment: performed, on average, at least once a year.

Comment: optimal follow-up frequency according to European guidelines:

- under 1 year of age — at least once every 2 months

- from 1 year to 12 years — at least once every 6 months

- from 12 to 18 years — at least once every 6 months

- over 18 years — at least once a year

- during pregnancy — at least once per trimester [16].

According to the Russian expert working group, it is advisable to conduct more frequent monitoring in patients under 1 year of age — at least once a month; from 1 year — at least once every 3 months; from 7 years — at least once every 6 months; over 18 years — at least once a year; and in pregnant women — at least once a month [16].

These recommendations are based on the years of experience of physicians specializing in the treatment of phenylketonuria patients in the Russian Federation and align with similar guidelines developed in EU countries [16].

Comments: Consultation with a clinical nutritionist is indicated for patients to provide specialized medical care. The pathogenetic treatment method for phenylketonuria is medical nutrition therapy. The frequency of visits to a clinical nutritionist is determined by clinical indications.

Comments: Appropriate programs and tests are used to assess intelligence and other mental activities in patients with HPA. During the transitional period, adolescents are in particular need of psychological assistance regarding family and social communication, as well as career guidance; for women with HPA, the focus should be on pregnancy-related issues.

Psychological assistance for patients may require a series of sessions, depending on the individual characteristics of the patient and family.

Comments: HPA is not a contraindication for prophylactic vaccination [140, 141]. In the presence of concomitant diseases, clearance for vaccination is provided by the relevant specialist. These recommendations are based, inter alia, on many years of experience of physicians specializing in the treatment of patients with phenylketonuria in the Russian Federation.

6. Organization of Medical Care

6.1 Indications for hospitalization

Indications for scheduled hospitalization in a medical organization:

1. Provision of diagnosis and treatment requiring prolonged (day or round-the-clock stay) medical observation and monitoring of clinical and laboratory parameters;

2. The need for various types of expert evaluations or examinations in a medical organization when they cannot be performed on an outpatient basis (including issuing a conclusion by a federal medical board / medical commission).

3. Sapropterin sensitivity testing**.

Comments: The sapropterin** loading test can be performed in either an inpatient or day-care hospital setting (Appendix A3.12).

During inpatient treatment, the patient is prescribed a low-phenylalanine diet and an amino acid mixture.

Indications for emergency hospitalization in a medical organization:

1. Acute conditions threatening life and the development of complications that require emergency treatment, including intensive care, as well as round-the-clock medical observation and special diagnostic and therapeutic procedures.

Indications for discharging a patient from a medical organization:

1. Absence of a threat to the patient's life and the threat of complications requiring emergency treatment upon completion of diagnostic measures.

2. Stabilization of the condition and key clinical and laboratory parameters of the pathological process, with no need for prolonged medical observation (day or round-the-clock stay);

3. Completion of the patient's diagnostic and treatment plan, with recommendations given for further observation and treatment.

4. The need to transfer the patient to another medical institution or social security institution.

6.2 Principles of organizing medical care for patients with hyperphenylalaninemia

Making a diagnosis of HPA raises many questions for the patient's relatives and for specialists working with such patients. Upon the detection and progression of the disease, numerous problems arise that require competent organization of care involving a multidisciplinary team of specialists and compliance with basic principles and approaches to its delivery.

These principles must be observed at any stage of medical care delivery, both at the time of diagnosis and at any stage of patient follow-up.

1) The patient and their representatives must receive complete information about the disease, its course, pathogenesis, prognosis, complications, and potential therapy methods.

2) A diagnosis of HPA implies the provision of primary, specialized, and palliative care at all stages of the disease.

3) Upon diagnosis of HPA, the family should be referred to a specialist with experience working with HPA patients, possessing up-to-date information on the course of the disease and opportunities for participation in clinical trials (of new drugs and/or medical devices). It is also important to inform families about existing patient advocacy organizations working with this patient group.

4) Monitoring of a patient with HPA should involve a multidisciplinary team of specialists consisting of: a pediatrician / general practitioner, medical geneticist, clinical nutritionist, neurologist, medical psychologist, endocrinologist / pediatric endocrinologist, psychiatrist, cardiologist / pediatric cardiologist, physical therapy physician / rehabilitation physician / physical and rehabilitation medicine physician, and gastroenterologist. Other specialists should be involved as specific problems arise.

Comments: The medical geneticist provides primary follow-up care for patients with HPA. The duties of the medical geneticist include examination and diagnosis, informing the family about the disease, drawing up a multidisciplinary monitoring plan, patient follow-up, assessing the dynamics and progression of the disease during regular examinations, and prescribing pathogenetic therapy. Upon reaching fertile age in patients with HPA, the duties of the medical geneticist include drawing up a preconception care plan to prevent maternal PKU, and testing the partner for heterozygous carrier status of pathogenic variants in the PAH gene (or other genes depending on the patient's form of HPA). When drawing up the multidisciplinary monitoring plan, the medical geneticist determines the frequency of follow-up by other specialists of the multidisciplinary team in accordance with the patient's current condition. At each examination, the medical geneticist reassesses the patient's current condition and accordingly adjusts the multidisciplinary monitoring plan. The medical geneticist also monitors a child with maternal PKU syndrome (consultation, determining the examination strategy, including the need for DNA diagnostics and interpretation of its results).

7. Additional Information

HPA is a chronic incurable disease, so patients need active dispensary follow-up and continuous treatment. With timely diagnosis and adequate therapy, the clinical picture is more favorable; without it, nervous system damage progresses rapidly. Many patients who are diagnosed in a timely manner and receive adequate therapy live to old age, live full lives, and have healthy offspring.

Criteria for Evaluating the Quality of Medical Care

Criteria for assessing the quality of medical care

Criteria for evaluating the quality of specialized medical care

References

Appendix A1. Working Group Composition

1. Inga V. Anisimova — Cand. Sci. (Med.), Medical Geneticist, Head of the Department of Medical Care Organization, Academician N.P. Bochkov Research Centre for Medical Genetics, Member of the Association of Medical Geneticists (AMG).

2. Elena G. Bakulina — Cand. Sci. (Med.), Deputy Chief Physician, Stavropol Krai Clinical Consultative and Diagnostic Center, Member of the Association of Medical Geneticists (AMG).

3. Alexander A. Baranov — Academician of the Russian Academy of Sciences, Professor, Dr. Sci. (Med.), Advisor to the Head of the Research Institute of Pediatrics and Child Health, Academician B.V. Petrovsky National Research Center of Surgery, Professor of the Department of Pediatrics and Pediatric Rheumatology, Sechenov First Moscow State Medical University of the Russian Ministry of Health (Sechenov University), Honorary President of the Union of Pediatricians of Russia.

4. Tatyana E. Borovik — Professor, Dr. Sci. (Med.), Head of the Laboratory of Nutrition for Healthy and Sick Children, National Medical Research Center for Children's Health of the Russian Ministry of Health, Member of the Association of Medical Geneticists (AMG).

5. Tatyana V. Bushuyeva — Dr. Sci. (Med.), Chief Researcher at the Laboratory of Nutrition for Healthy and Sick Children, National Medical Research Center for Children's Health of the Russian Ministry of Health, Member of the Association of Medical Geneticists (AMG).

6. Zulfiya I. Vafina — Head of the Medical Genetic Consultation at the Republican Clinical Hospital of the Ministry of Health of the Republic of Tatarstan, member of the Association of Medical Geneticists (AMG).

7. Elena A. Vishnyova — MD, Professor of the Russian Academy of Sciences, Deputy Director for Research at the Research Institute of Pediatrics and Child Health, National Medical Research Center No. 2 of the Petrovsky National Research Center of Surgery, Professor at the Department of Faculty Pediatrics, Pediatric Faculty, Pirogov Russian National Research Medical University of the Russian Ministry of Health, member of the Union of Pediatricers of Russia.

8. Tatiana A. Golikhina — PhD, Medical Geneticist at the Research Institute - Regional Clinical Hospital No. 1 named after Professor S.V. Ochapovsky of the Ministry of Health of Krasnodar Krai, member of the Association of Medical Geneticists (AMG).

9. Polina Gundorova — PhD, Researcher at the DNA Diagnostics Laboratory, Research Centre for Medical Genetics named after Academician N.P. Bochkov.

10. Nataliya V. Zhurkova — PhD, Leading Researcher at the Research Institute of Pediatrics and Child Health, National Medical Research Center No. 2 of the Petrovsky National Research Center of Surgery, member of the Union of Pediatricers of Russia, member of the Association of Medical Geneticists (AMG).

11. Ekaterina Yu. Zakharova — MD, Head of the Laboratory of Hereditary Metabolic Diseases, Research Centre for Medical Genetics named after Academician N.P. Bochkov, member of the Russian Society of Medical Geneticists, member of the Society for the Study of Inborn Errors of Metabolism (SSIEM).

12. Natalia G. Zvonkova — PhD, Senior Researcher at the Laboratory of Nutrition for Healthy and Sick Children, National Medical Research Center for Children's Health of the Russian Ministry of Health, member of the Association of Medical Geneticists (AMG).

13. Lyudmila M. Kuzenkova — Professor, MD, Head of the Center of Pediatric Psychoneurology, Head of the Department of Psychoneurology and Psychosomatic Pathology, National Medical Research Center for Children's Health of the Russian Ministry of Health, member of the Association of Medical Geneticists (AMG).

14. Sergey I. Kutsev — Academician of the Russian Academy of Sciences, Professor, MD, Director of the Research Centre for Medical Genetics named after Academician N.P. Bochkov, President of the Association of Medical Geneticists (AMG).

15. Artur Sh. Latypov — PhD, Head of the Medical Genetic Center at the Moscow Regional Research Clinical Institute (MONIKI), member of the Association of Medical Geneticists (AMG).

16. Lidiya V. Lyazina — PhD, Medical Geneticist, Saint Petersburg State Healthcare Institution "Diagnostic Center (Medical Genetic)", member of the Association of Medical Geneticists (AMG).

17. Svetlana A. Matulevich — MD, Head of the Medical Genetic Consultation at the Research Institute - Regional Clinical Hospital No. 1 named after Professor S.V. Ochapovsky of the Ministry of Health of Krasnodar Krai, member of the Association of Medical Geneticists (AMG).

18. Lyudmila P. Nazarenko — Professor, MD, Deputy Director for Research and Clinical Work, Head of the Laboratory of Hereditary Pathology at the Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, member of the Association of Medical Geneticists.

19. Leyla S. Namazova-Baranova — Academician of the Russian Academy of Sciences, Professor, MD, Head of the Research Institute of Pediatrics and Child Health, National Medical Research Center No. 2 of the Petrovsky National Research Center of Surgery, Head of the Department of Faculty Pediatrics, Pediatric Faculty, Pirogov Russian National Research Medical University of the Russian Ministry of Health, President of the Union of Pediatricers of Russia.

20. Natalia V. Nikitina — PhD, Medical Geneticist at the Medical Genetic Consultation of the Clinical and Diagnostic Center "Maternal and Child Health Protection", member of the Association of Medical Geneticists.

21. Ekaterina A. Nikolaeva — MD, Chief Researcher at the Department of Clinical Genetics, Veltischev Research and Clinical Institute of Pediatrics, Professor at the Department of Innovative Pediatrics and Pediatric Surgery, Institute of Continuing Education and Professional Development, Pirogov Russian National Research Medical University of the Russian Ministry of Health, member of the Association of Medical Geneticists.

22. Aleksandr V. Polyakov — Corresponding Member of the Russian Academy of Sciences, Professor, MD, Head of the DNA Diagnostics Laboratory, Research Centre for Medical Genetics named after Academician N.P. Bochkov, Head of the Department of Molecular Genetics and Bioinformatics, Institute of Higher and Continuing Professional Education of the Research Centre for Medical Genetics, member of the Association of Medical Geneticists.

23. Aleksandr A. Pushkov — PhD, Leading Researcher at the Laboratory of Molecular Genetics and Cell Biology, National Medical Research Center for Children's Health of the Russian Ministry of Health, member of the Association of Medical Geneticists.

24. Svetlana A. Repina — PhD, Medical Geneticist at the Department of Medical Care Organization, Research Centre for Medical Genetics named after Academician N.P. Bochkov, member of the Association of Medical Geneticists.

25. Kirill V. Savostyanov — PhD, Head of the Center for Fundamental Research in Pediatrics, National Medical Research Center for Children's Health of the Russian Ministry of Health, member of the Association of Medical Geneticists.

26. Gulnara N. Seitova — PhD, Chief Physician of the Medical Genetic Center (Genetic Clinic), Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences.

27. Liliya R. Selimzyanova — PhD, Head of the Department of Standardization and Study of Evidence-Based Medicine Foundations, Research Institute of Pediatrics and Child Health, National Medical Research Center No. 2 of the Petrovsky National Research Center of Surgery, Associate Professor at the Department of Faculty Pediatrics, Pediatric Faculty, Pirogov Russian National Research Medical University of the Russian Ministry of Health, Associate Professor at the Department of Pediatrics and Pediatric Rheumatology, I.M. Sechenov First Moscow State Medical University of the Russian Ministry of Health (Sechenov University), member of the Union of Pediatricers of Russia.

28. Natalia A. Semyonova — PhD, Senior Researcher at the Scientific Advisory Department, Research Centre for Medical Genetics named after Academician N.P. Bochkov, member of the Association of Medical Geneticists (AMG).

29. Olga Ya. Smirnova — Medical Geneticist, Senior Researcher at the Department of Standardization and Study of Evidence-Based Medicine Foundations, Research Institute of Pediatrics and Child Health, National Medical Research Center No. 2 of the Petrovsky National Research Center of Surgery, member of the Union of Pediatricers of Russia.

30. Dmitry M. Subbotin — Medical Geneticist at the Research Centre for Medical Genetics named after Academician N.P. Bochkov, member of the Association of Medical Geneticists (AMG).

31. Maria G. Sumina — Head of the Medical Genetic Counseling Department, Medical Geneticist, Sverdlovsk Region Clinical and Diagnostic Center "Maternal and Child Health Protection".

32. Andrey P. Fisenko — Professor, MD, Director of the National Medical Research Center for Children's Health of the Russian Ministry of Health, President of the Professional Public Organization (Association) of Pediatric Doctors "Initiative of Pediatric and Neonatal Specialists in the Development of Clinical Practices" on pediatric issues.

33. Tatiana V. Fedotova — PhD, Head of the Medical Genetic Consultation at the Voronezh Regional Clinical Hospital No. 1, member of the Association of Medical Geneticists (AMG).

34. Elena A. Shestopalova — PhD, Head of the Moscow Neonatal Screening Center (Medical Genetic Department) at the Morozov Children's City Clinical Hospital of the Moscow Healthcare Department, member of the Association of Medical Geneticists (AMG).

    Representatives of public organizations:

35. Elena N. Balabanova — President of the ANO "Phenylketonuria Patients Society", Moscow

36. Olga A. Bashkirova — Chairperson of the Regional Public Organization of the Republic of Tatarstan "Support for Patients with Phenylketonuria"

37. Natalia I. Marechenkova — Chairperson of the Kemerovo Regional Public Organization of Parents of Children Diagnosed with Phenylketonuria

38. Victoria A. Sverdlova — Chairperson of the Novosibirsk Regional Public Organization of Invalids with Phenylketonuria and Other Rare Diseases "Formula of Life"

39. Tatiana A. Sleptsova — Director of the Regional Public Organization for Assistance to People with Phenylketonuria of the Saratov Region "Taste of Life"

40. Matlyuba A. Khakimova — Chairperson of the Board of the Regional Public Organization for Assistance to People with Phenylketonuria and Other Rare Diseases "PKU RB" of the Republic of Bashkortostan, Head of the PKU Division at the All-Russian Society of Rare (Orphan) Diseases.

    The authors declare no financial support or conflicts of interest that need to be disclosed.

Appendix A2. Methodology for Developing Clinical Guidelines

These guidelines are intended for use by medical organizations and institutions of federal, territorial, and municipal healthcare management systems, compulsory and voluntary medical insurance systems, and other medical organizations of various legal forms engaged in the provision of medical care.

These guidelines establish the types, scope, and quality indicators of medical care for patients with phenylketonuria and were reviewed on October 23, 2019, at an expert council meeting in Moscow.

The clinical guidelines were developed based on a literature review of 1990–2023 from the Medline (Pubmed version), Embase (Dialog version), and Cochrane Library databases, Orphanet and OMIM, as well as international clinical guidelines for the diagnosis, treatment, and management of patients with metabolic diseases.

Internet data presented on international and national websites of rare disease societies and public organizations were used. The search depth was 20 years.

The evaluation of the quality of evidence and the strength of recommendations for the application of medical technologies was performed in accordance with the standardized scale presented in Tables 1–3.

Target audience for these clinical guidelines:

1. Medical geneticists;

2. General practitioners (family physicians);

3. Pediatricians;

4. General practitioners;

5. Dietitians;

6. Neurologists;

7. Psychiatrists;

8. Endocrinologists;

9. Pediatric endocrinologists;

10. Cardiologists;

11. Pediatric cardiologists

12. Medical psychologists;

13. Obstetricians-gynecologists;

14. Neonatologists;

15. Laboratory geneticists;

16. Clinical laboratory diagnostics physicians;

17. Medical students;

18. Residents. 

Table 1. Level of Evidence (LoE) grading scale for diagnostic methods (diagnostic interventions)

Table 2. Scale for assessing levels of reliable evidence (LRE) for prevention, treatment, and rehabilitation methods (preventive, therapeutic, rehabilitation interventions)

Table 3. Grade of Recommendation (GoR) scale for prevention, diagnostic, treatment, and rehabilitation methods (preventive, diagnostic, therapeutic, and rehabilitative interventions)

Procedure for updating clinical guidelines.

The mechanism for updating the clinical guidelines provides for their systematic revision at least once every three years, as well as upon the emergence of new evidence-based medicine data regarding the diagnosis, treatment, prevention, and rehabilitation of specific diseases, or in the presence of justified additions/comments to the previously approved clinical guidelines, but no more frequently than once every 6 months.

Appendix A3. Reference Materials

Appendix A3.1 Main regulatory legal acts

1. Federal Law No. 323-FZ of November 21, 2011, "On the Fundamentals of Healthcare Protection in the Russian Federation".

2. Decree of the Government of the Russian Federation No. 890 of July 30, 1994, "On State Support for the Development of the Medical Industry and Improvement of the Provision of Medications and Medical Devices to the Population and Healthcare Institutions" (with amendments and addenda)

3. Order of the Russian Ministry of Health No. 274n of April 21, 2022, "On Approval of the Procedure for Providing Medical Care to Patients with Congenital and (or) Hereditary Diseases".

4. Government Decree No. 403 of April 26, 2012, "On the Procedure for Maintaining the Federal Register of Persons Suffering from Life-Threatening and Chronically Progressive Rare Orphan Diseases Leading to a Reduction in Citizens' Life Expectancy or Disability, and Its Regional Segment".

5. Criteria for Assessing the Quality of Medical Care (Order of the Ministry of Health of the Russian Federation No. 203n of May 10, 2017, "On Approval of Criteria for Assessing the Quality of Medical Care").

6. Order of the Ministry of Health of the Russian Federation No. 834n of December 15, 2014, "On Approval of Unified Forms of Medical Documentation Used by Medical Organizations Providing Outpatient Medical Care and Procedures for Their Completion".

7. Order of the Russian Ministry of Health No. 1051n of November 12, 2021, "On Approval of the Procedure for Giving Informed Voluntary Consent to Medical Intervention and Refusal of Medical Intervention, the Form of Informed Voluntary Consent to Medical Intervention, and the Form of Refusal of Medical Intervention" (along with the "Procedure for Giving Informed Voluntary Consent to Medical Intervention and Refusal of Medical Intervention Regarding Certain Types of Medical Interventions")

8. Order of the Russian Ministry of Health No. 1094n of November 24, 2021, "On Approval of the Procedure for Prescribing Medicinal Products, Prescription Form Templates for Medicinal Products, the Procedure for Issuing These Forms, Their Accounting and Storage, Prescription Form Templates Containing Prescriptions for Narcotic Drugs or Psychotropic Substances, the Procedure for Their Manufacture, Distribution, Registration, Accounting, and Storage, as Well as the Rules for Issuing Prescription Forms, Including in the Form of Electronic Documents"

9. Government Decree No. 333 of April 9, 2015, "On Approval of the Rules for Generating the List of Specialized Medical Nutrition Products for Disabled Children"

Drug information: https://grls.rosminzdrav.ru/

Main regulatory legal acts governing the provision of palliative medical care

1. Federal Law No. 18-FZ dated March 6, 2019, "On Amendments to the Federal Law 'On the Basics of Protecting the Health of Citizens in the Russian Federation' Regarding the Provision of Palliative Medical Care."

2. Order of the Russian Ministry of Health No. 345n, Russian Ministry of Labor No. 372n dated May 31, 2019, "On approval of the Regulations on the organization of palliative medical care, including the procedure for interaction between medical organizations, social service organizations, and public associations, and other non-profit organizations operating in the field of health protection."

3. Order of the Russian Ministry of Health No. 348n dated May 31, 2019, "On approval of the list of medical devices intended to maintain human organs and body systems, provided for home use."

4. Order of the Russian Ministry of Health No. 505n dated July 10, 2019, "On approval of the Procedure for the transfer by a medical organization to a patient (their legal representative) of medical devices intended to maintain the functions of human organs and body systems for home use in the provision of palliative medical care."

Other regulatory and legal documents used in the development of the clinical guidelines:

1. International Statistical Classification of Diseases and Related Health Problems (ICD-10);

2. Order of the Russian Ministry of Health No. 205n of May 2, 2023, "On Approval of the Nomenclature of Positions for Medical Workers and Pharmaceutical Workers"

3. Order of the Ministry of Health of the Russian Federation No. 103n dated February 28, 2019, "On Approval of the Procedure and Timeframes for the Development and Revision of Clinical Guidelines, Their Template Form, and Requirements for Their Structure, Composition, and Scientific Validity of Information Included in Clinical Guidelines".

4. Order of the Russian Ministry of Health No. 804n dated October 13, 2017, "On approval of the nomenclature of medical services."

5. Order of the Ministry of Health and Social Development of the Russian Federation dated August 9, 2005, No. 494, "On the procedure for the use of medicinal products in patients for vital indications"

Appendix A3.2 Etiopathogenetic classification of hyperphenylalaninemia

*http://www.ncbi.nlm.nih.gov/omim/?term=HPA Blau monograph

Appendix A3.3 Diagnostic indicators of phenylalanine and pterins in blood, urine, and cerebrospinal fluid

* AdGTPCH – autosomal dominant guanosine triphosphate cyclohydrolase I deficiency; ArGTPCH – autosomal recessive guanosine triphosphate cyclohydrolase I deficiency, PTPS – 6-pyruvoyltetrahydropterin synthase, SR – sepiapterin reductase, PCD – pterin-4-alpha-carbinolamine dehydratase, DHPR – dihydropteridine reductase, Phe – phenylalanine, Neo – neopterin, Bio – biopterin, Pri – primapterin, 5-HIAA – 5-hydroxyindoleacetic acid, HVA – homovanillic acid, 5-MTHF – 5-methyltetrahydrofolate, B – blood, U – urine, CSF – cerebrospinal fluid.

Appendix A3.4 Algorithm for molecular genetic diagnostics of patients with hyperphenylalaninemia

Search for PAH gene mutations

Comment: Regardless of the diagnostic blood Phe concentration, the most frequent (98%) cause of all HPA is mutations in the PAH gene. Searching for mutations in the PAH gene is a molecular genetic test that should be ordered first. An exception is patients who have undergone urine or blood pterin testing, the results of which clearly suggest BH₄-deficient HPA.

1. Search for common PAH gene mutations

Investigation of the coding sequence of the PAH gene (Sanger sequencing or next-generation sequencing followed by confirmation of results using Sanger sequencing)

2. Search for PAH gene deletions and duplications (MLPA assay)

Comment: If one or both PAH gene mutations are not detected after searching for common mutations (a), tests (b) and (c) are successively ordered. If both mutations are not detected as a result of complete PAH gene analysis, gene testing should be performed according to the algorithm below.

3. Search for mutations in PTS, QDPR, GCH1, PCBD1, SPR, and DNAJC12 genes

Comment: If biochemical test results are available confirming a specific form of BH₄-deficient HPA in the patient, one can proceed directly to testing the corresponding gene. Otherwise, it is recommended to test the genes in the specified order: PTS, QDPR, GCH1, PCBD1, SPR, DNAJC12 (by frequency of occurrence). When using next-generation sequencing, it is possible to test the above genes simultaneously. It should be noted that all results obtained using next-generation sequencing methods cannot be used in clinical practice without confirmation by a reference method (Sanger sequencing for single nucleotide variants), as well as family segregation analysis of the identified variants (determining cis- or trans- position for autosomal recessive diseases).

Appendix A3.5 Informed consent of the legal representative of a child with HPA regarding the need for treatment and compliance with the doctor's requirements

I, __________________________________________________________________________

(Full name)

am the legal representative (mother, father, adoptive parent, guardian, trustee) of the child or person recognized as legally incompetent

_____________________________________________________________________________

(Full name of the child or incompetent citizen, year of birth)

am aware of the need to comply with the doctor's requirements and treatment.

I have been provided with complete and comprehensive information regarding the nature and severity of the disease, potential complications, including data on examination results, prognosis, possible options for medical intervention (examination, treatment), and expected outcomes of the ongoing treatment.

The possible consequences of non-compliance with the physician's instructions and treatment (such as mental retardation, epileptic seizures, etc.) have been explained to me in detail in an accessible form. I am aware that failure to follow the physician's recommendations and treatment plan will negatively affect the child's health and may even lead to an adverse outcome.

___________ ___________________________________________________ «___»____20__

   (signature)           (Full name of the citizen or their legal representative)

___________ ___________________________________________________ «___»____20__

(signature)                   (Position and Full name of the medical worker)

Appendix A3.6 Chemical composition and age of administration of specialized amino acid-based products without phenylalanine

(per 100 g of dry product / 100 ml of ready-to-use liquid product) [16, 25, 27, 31, 53]

Appendix A3.7 List of low-protein and protein-free specialized products registered in the Russian Federation

Appendix A3.8 Physiological requirement norms for basic nutrients in healthy children of various age groups and adults

Average daily requirements for basic nutrients and energy for infants in the first year of life (per kg of body weight) [24].

Average daily norms of physiological requirements for basic nutrients and energy for children over one year of age [24].

Appendix A3.9 Minimum permissible amount of Phe in the diet of patients with HPA depending on age

Note: *- [23]; ** — [4, 20].

The missing amount of protein is replenished using specialized medical foods ― Phe-free amino acid mixtures and low-protein foods. Amino acid mixtures vary in protein content (from 13 g to 77.5 g per 100 g of dry product) and other nutrients (carbohydrates, fats, vitamins, micro- and macronutrients). All mixtures are Phe-free. Amino acid mixtures containing 13–15 g of protein per 100 g of dry mixture are intended for infants in the first year of life. For children over 1 year of age, adolescents, and adults, including pregnant women, mixtures with a higher protein content are prescribed (Appendix A3.3). The calculation of the daily dose of the specialized product is carried out using the formula:

where Ps is the daily protein intake, Pn is the protein from natural foods, and P is the protein equivalent in 100 g of the dry specialized product. The calculation principle for a low-phenylalanine diet is demonstrated using the dietary calculation for a 3-year-old child with PAH-dependent HPA (PKU).

Appendix A3.10 Example of nutrition calculation for a child with phenylalanine hydroxylase-dependent hyperphenylalaninemia

Patient age 3 years, body weight 14.5 kg

1. Total daily protein intake in the patient's diet (see Appendix A3.8): 54.0 g

2. Permissible daily Phe intake (see Appendix A3.9):

25 × 14.5 = 363 mg

3. Permissible amount of protein from natural foods (1 g of protein contains 50 mg of Phe):

363 : 50 = 7.3 g

4. Amount of protein provided by the specialized product based on a Phe-free L-amino acid mixture:

54.0 - 7.3 = 46.7 g

5. Daily amount of the amino acid mixture (100 g contains 20 g of protein):

46.7 × 100 : 20 = 233 g

6. Recommended daily fat intake in the diet (see Appendix A3.8): 60 g

7. Recommended daily carbohydrate intake (see Appendix A3.8): 261 g

Appendix A3.11 Treatment regimen for various BH4-deficient conditions

Treatment regimens for various BH4-deficient states (with/without hyperphenylalaninemia) *

Note: *-table compiled according to N. Blau, 2014.

Appendix A3.12 Sapropterin responsiveness test**

Testing for potential responsiveness to sapropterin** and subsequent treatment with it are carried out and monitored by the physician who manages patients with HPA. For closer monitoring, it is advisable to perform the test in an inpatient setting, possibly a day-care unit, or outpatiently if the patient's compliance is satisfactory. Before testing, informed consent (Appendix A3.13) for the procedure is obtained from the parents (legal representatives) of the child, as well as from patients older than 14 years, and a preliminary discussion is held to explain the goals and objectives of the testing and the role of the parents and the patient in ensuring and complying with the test conditions.

Important testing conditions include:

- maintaining a food diary and a stable low-phenylalanine diet to exclude its influence on fluctuations in blood Phe concentration;

- the need for close interaction with the physician to promptly report adverse events, intercurrent illnesses, or other events that could affect the test result;

- blood samples for Phe determination should be collected immediately before sapropterin** administration, and then 2-5 samples during the trial period at the same time of day, preferably on an empty stomach;

- measuring Phe in dried blood spots using the same method.

Testing can be performed starting from 1 year of age to identify BH4-deficient forms of HPA, which does not preclude repeating the test at an older age to determine responsiveness to sapropterin** in patients with a PAH-dependent form of HPA.

Various testing protocols can be used (short protocols: 24 hours, 48 hours, 7 days; long protocols: 28 days or more). The duration of testing and the frequency of blood sampling during testing are determined by the physician, guided by research data on the optimal performance of the test [28].

To obtain the most informative result, it is advisable to use the maximum dosage of #sapropterin** (20 mg/kg) for testing [5-7]. The daily dose is calculated individually for each patient in accordance with the instructions.

"Short" testing protocols

The main criterion for drug efficacy in short testing is a decrease in blood Phe concentration by 30% or more.

Performing the test in children over 1 year of age, adolescents, and adult patients

Prior to testing, blood Phe levels are stabilized by maintaining a constant dietary intake for 4-5 days before the start of the test and during the testing period. Testing begins against the background of a stable blood Phe level, while calculating the average value of two Phe measurements obtained from double blood sampling before the start of testing. It is advisable that the average Phe value is not lower than 450 µmol/L (7.5 mg/dL); therefore, "short" testing should be performed primarily in patients with elevated blood Phe levels. If blood Phe levels are normal, the diet may be slightly expanded (only with the consent of the parents/legal representatives or the patient) until stable blood Phe values of at least 450 µmol/L (7.5 mg/dL) are achieved.

On the first day of drug administration on an empty stomach, a capillary blood sample for phenylalanine is taken, then the child takes the calculated dose of the drug with breakfast or immediately after breakfast. For 2 (or 7) days in the morning at the same time after breakfast (or with a meal), the child takes the drug dissolved in warm water in accordance with the instructions.

Repeat blood sampling is performed on the 3rd (or 8th) day strictly on an empty stomach. Response to drug sensitivity is evaluated by the degree of decrease in the patient's blood Phe concentration while maintaining a stable low-phenylalanine diet. A patient is considered responsive if the difference between the Phe level obtained at the end of the treatment response evaluation period and the baseline Phe level before starting the drug is 30% or more.

If the Phe level decreases by more than 85%, there is a high probability that the patient's hyperphenylalaninemia is caused by tetrahydrobiopterin (BH4) deficiency.

Sample testing protocols are shown in Tables 1-2.

Table 1. Two-day testing protocol to determine sensitivity to sapropterin*.

*- the frequency of blood sampling can be increased up to 3 times a day at the physician's discretion. More frequent Phe monitoring makes it possible to capture the moment of maximum reduction in blood Phe concentration during sapropterin** administration.

Table 2. Seven-day testing protocol to determine sensitivity to sapropterin**¹.

¹- the frequency of blood sampling can be increased up to 3 times a day at the physician's discretion. More frequent Phe monitoring makes it possible to capture the moment of maximum reduction in blood Phe concentration during sapropterin** administration.

After completing testing and obtaining a positive result, it must be confirmed during long-term follow-up.

"Long-term" test

The main criterion for the efficacy of "long-term" testing is an increase in tolerance to dietary phenylalanine. The advantage of such testing is the possibility of performing it against the background of the patient's reference blood Phe values (i.e., not higher than 360 µmol/L). This requires daily maintenance of a food diary and careful control of the diet, the patient's actual intake of natural protein and Phe.

During the long-term sapropterin** response test, blood samples for Phe concentrations should be collected daily if possible; if not possible, at least once a week.

Maximum tolerance to dietary Phe is determined by gradually increasing Phe intake (diet expansion) while maintaining the patient's blood Phe levels within the target range. In accordance with European guidelines for the diagnosis and management of HPA, an increase in natural protein tolerance of ≥100% compared to baseline (before testing) while maintaining blood Phe concentrations within the therapeutic range after 6 months of sapropterin** administration is proof of the patient's responsiveness to treatment with this drug.

During the trial treatment and subsequent therapy adjustment, the blood Phe concentration must not exceed the target value of 6 mg/dL (360 µmol/L). When combining a low-phenylalanine diet and sapropterin**, anthropometric and biochemical parameters of nutritional status must be systematically monitored to prevent the development of major and minor nutrient deficiencies and nutrition-related conditions.

If necessary, long-term pharmacotherapy in HPA patients who respond to sapropterin** treatment with a decrease in blood Phe levels is carried out in combination with a diet using amino acid supplements, the amount of which is determined by the physician.

Patients receiving sapropterin** require careful monitoring of their nutritional status and other health parameters at least once every 6 months.

Testing for sensitivity to sapropterin** during pregnancy.

Female patients with HPA are strongly recommended to undergo testing before pregnancy. The possibility of performing a 48-hour sapropterin** sensitivity test is considered in cases where a pregnant woman with HPA cannot achieve a blood Phe level of ≤360 µmol/L using a low-phenylalanine diet. This approach is considered justified, as the risk of teratogenicity from high maternal blood concentrations outweighs the risk of using sapropterin** during pregnancy. If testing was performed previously (in adolescence, before pregnancy, etc.) and a positive response was obtained, allowing the HPA woman to expand her diet, the drug is prescribed during the preconception period (2-3 months before the planned conception). After pregnancy occurs, administration of the drug is continued until delivery, with dose adjustment of the medicinal product [26, 54].

Pharmacotherapy for BH4-deficient conditions with HPA is pathogenetic and is prescribed obligatorily after establishing the diagnosis without testing. The initial dose of sapropterin** in patients with BH₄ deficiency ranges from 2 to 5 mg/kg of body weight administered once daily. If necessary, the dose of sapropterin** can be increased to 20 mg/kg of body weight per day; to achieve an optimal therapeutic effect, administration 2-3 times a day is possible.

Appendix A3.13 Informed consent for testing with sapropterin**

I, _______________________________________________________________________ (Full Name),

passport No. ____________, issued by ___________________________________________________

am informed that the medicinal product sapropterin** (sapropterin dihydrochloride) is a synthetic analogue of natural tetrahydrobiopterin and is a treatment for patients with inherited metabolic disorders (underline as applicable) – phenylketonuria responsive to sapropterin** treatment, and tetrahydrobiopterin metabolism disorders (atypical, cofactor, tetrahydrobiopterin-dependent form of phenylketonuria), from which my child (Full Name) _______________________________________________________________, born in ____________, is suffering.

I am informed that to confirm sensitivity to sapropterin** treatment, testing must be performed, the purpose of which is to confirm a decrease in blood phenylalanine levels during sapropterin** administration in accordance with available international standards (30% or more).

I am informed that in case of confirmed sensitivity to sapropterin** therapy, such treatment will be provided to my child only after confirmation of state funding.

I am informed by the attending physician that side effects may occur during the use of sapropterin**, the most frequent of which are:

• Mild headache

• Rhinitis, nasal congestion

• Sore throat

• Cough

• Gastrointestinal disorders: diarrhea, vomiting, abdominal pain

• Hypophenylalaninemia

• Other side effects not yet known may occur.

I undertake to provide my child with a stable diet and make no changes to it during the testing period.

I undertake to inform the attending physician about all side effects, adverse events, and changes in my child's condition that develop during the use of sapropterin**.

I am informed that I have the right to refuse treatment with the drug at any time, and I undertake to notify the attending physician within 24 hours.

I am informed about the recommended dose of the drug, treatment regimen, and expected therapeutic outcomes.

My signature under this consent signifies that the text of this consent has been read, is fully understood, I have received satisfactory answers to all my questions, and I agree with the proposed treatment plan.

___________________________

(date: day, month, year)

Legal representative (mother/father/other relative)

______________________________________ (signature)

______________________________________ (Full Name)

Attending physician __________________________ (signature)

______________________________________ (Full Name)

Healthcare facility: ____________________________________________

Appendix A3.14 Collection of biomaterial for diagnostics in blood spots

Preparation for blood collection

Preparation for blood collection is standard: medical personnel must observe sterility rules by wiping the puncture site with an antiseptic solution and using disposable lancets and gloves.

Obtaining a dried blood spot

As a rule, for obtaining dried blood spots [72], blood is collected from a finger; venous blood collected from an EDTA-containing tube may also be used (depending on the test kits used).

In newborns, blood for neonatal screening and retesting is collected exclusively from the heel, and in exceptional cases from a finger (if heel blood collection is impossible).

The puncture site (heel, finger) should be treated with a sterile wipe soaked in 70% alcohol, then blotted with a dry sterile wipe. The first drop formed at the puncture site is not used — it is wiped away with a dry cotton swab. Subsequent drops are applied consecutively into the circles on the absorbent test form. The circles on the form must be saturated evenly and completely, with no white spots on the reverse side of the form (Fig. 1G3). Obtaining a single spot requires 80–100 µL of blood. After applying blood to the test form, the sample is allowed to dry completely for at least 2 hours at room temperature, without contacting other test forms. After drying, the test forms are placed in individual packaging without touching or overlapping blood spots.

Figure 1G3. Dried blood spots on a test form.

For molecular genetic and biochemical studies outside of neonatal screening, in addition to droplet application, the required volume of whole blood may be applied to the test form using a semi-automatic dispenser, for example, from a tube containing the analyzed biological material.

Transportation of test forms is carried out at a temperature not exceeding 25°C [MR SPK].

Algorithm of actions for medical personnel during blood sample collection:

● wash hands (hygienic level), put on gloves;

● wash the patient's hands (the child's heel if blood is collected from the heel);

● wipe the puncture area with a sterile wipe soaked in 70% alcohol, blot with a dry sterile wipe; puncture with a sterile disposable lancet;

● wipe away the first drop of blood with a sterile dry swab;

● apply gentle pressure to obtain a second drop of blood;

● apply the test form perpendicularly to the blood drop and saturate it completely with blood;

● similarly apply 6–8 drops to the test form; the appearance of the blood spots must be identical on both sides;

● dry the test form in a horizontal position on a clean, degreased surface for at least 4 hours without heat treatment and direct sunlight;

● pack the test forms in a clean envelope so that the blood spots do not touch.

Specifics during infusion therapy

Certain patients may receive infusion therapy or blood component transfusions, which can affect test results. For example, plasma transfusions may yield false-negative results because the enzymes being determined are present in both plasma and blood cells. It is recommended to perform blood collection for enzyme and DNA diagnostics no earlier than 6–7 days after plasma transfusion and 7–10 days after blood component transfusion.

Blood collection is not permitted:

· immediately after infusion therapy (can be done 48–72 hours later);

· immediately after exchange transfusion (can be done 120 days after exchange transfusion).

Storage and transportation of biological material

Dried blood spot samples can be stored in a standard refrigerator compartment at +4°C until shipment. The storage period prior to shipment must not exceed 7 days. The temperature during transportation of the biological material must be between 0°C and +10°C. If stored longer and at a higher temperature, enzyme activity may decrease even in normal ranges, leading to false-positive results.

Algorithm of actions for medical personnel during blood sample collection

· wash hands (hygienic level), put on gloves;

· wash the patient's hands (the child's heel if blood is being drawn from the heel); it is important to warm the puncture site by wrapping it in a tissue soaked in warm water; the child should be in a vertical position;

· wipe the puncture site with a sterile gauze pad moistened with 70% alcohol, blot with a dry sterile gauze pad; press the limb against a firm surface, puncture with a sterile disposable lancet;

· wipe away the first drop of blood with a sterile dry swab;

· apply gentle pressure to obtain a second drop of blood;

· apply the test card perpendicularly to the drop of blood and saturate it completely with blood;

· similarly, apply 6-8 drops of blood to the test card; the blood spots must look identical on both sides.

· dry the test card in a horizontal position on a clean, grease-free surface for at least 4 hours without heat treatment or exposure to direct sunlight;

· pack the test cards in a clean envelope so that the blood spots do not touch.

Specifics during infusion therapy

Some patients may receive infusion therapy or blood component transfusions, which can affect test results. For example, blood plasma transfusions may yield false-negative results. It is recommended to perform blood collection for metabolite analysis and DNA diagnostics no earlier than 6-7 days after a blood plasma transfusion and 7-10 days after a blood component transfusion.

Blood collection is not permitted:

· immediately after infusion therapy (can be done 48–72 hours later);

· immediately after exchange transfusion (can be done 120 days after exchange transfusion).

Storage and transportation of biological material

Dried blood spot samples can be stored at room temperature and a humidity of no more than 60%. The storage period before shipment must not exceed 7 days.

Appendix B. Physician Action Algorithms

I General algorithm for diagnosis and therapy of HFA

II Algorithm for differential diagnosis of HFA

¹⁾Loading test is performed after 1 year

III Neonatal screening algorithm

Appendix C. Patient Information

What is phenylketonuria?

Phenylketonuria (PKU) is an inherited disorder characterized by impaired phenylalanine metabolism (Phe, an essential amino acid that is not synthesized in the body and comes from food ― animal and plant products, including breast milk and infant formula). PKU is a whole group of diseases of varying severity. In Russia, more than 200 children are born with this disease each year (approximately 1 in 7,000–8,000 newborns).

In the absence of timely diagnosis and treatment, the disease usually manifests at the age of 2–6 months with signs of central nervous system damage: parents are concerned by the child's lethargy, lack of interest in surroundings, sometimes increased irritability, anxiety, regurgitation, muscle tone disorders (more often muscle hypotonia), signs of atopic dermatitis, psychomotor development delay, and sometimes seizures. As they grow older, children suffer from severe nervous system damage up to mental retardation and epilepsy. With timely initiated pathogenetic treatment, symptoms are milder or absent.

How do doctors find out that a child is sick?

For early diagnosis and prevention of these symptoms, all newborns undergo neonatal screening in the maternity hospital on the 3rd–7th day of life.

The results of this examination are communicated to the parents by phone.

Parents must take the information received from the neonatal screening center seriously and immediately visit a doctor and/or contact them by phone, even if the child has no clinical symptoms. The decision on prescribing and the nature of diet therapy is made by the physician.

What problems does phenylketonuria cause?

The food we eat contains proteins made up of individual amino acids. In phenylketonuria, one of these amino acids, called phenylalanine, is not processed by the body in the usual way. Because of this, Phe accumulates in the child's blood. Without treatment, this leads to impaired brain development. However, this can be prevented by reducing the amount of protein in the diet. Thus, your child needs a special low-protein diet.

The diet, on the one hand, must be low in Phe, but on the other hand, it must be balanced to ensure the baby's growth and development. Your medical geneticist will help you better understand the situation so that it is completely clear to you.

What should parents do?

Except for the diet, treat the child as healthy; do not overprotect them.

The diet may seem difficult at first, but medical geneticists and dietitians will provide you with maximum support and help whenever you need it.

Will the child grow and develop normally?

For all children with PKU who started treatment on time, the answer is YES! If your child's treatment is carried out consistently, the child grows and develops normally.

However, if dietary compliance is insufficiently monitored, it can cause learning and behavioral problems in the child. The child will not be able to achieve the success they would have achieved with strict dietary compliance.

Is this a lifelong diet?

It is currently believed that lifelong dietary compliance is most favorable. Strict adherence to the diet is necessary for women with PKU immediately before and during pregnancy to prevent fetal malformations. Diet therapy based on a drastic restriction of Phe in the diet of sick children by excluding high-protein foods must be started no later than the first weeks of the child's life in order to achieve maximum treatment efficacy. The missing amount of protein is replenished with specialized therapeutic products partially or completely devoid of Phe [23].

The prescription of pathogenetic diet therapy from the first days of the child's life determines a favorable prognosis for the course of phenylketonuria.

Basic principles of the diet in PKU.

Regular food intended for a healthy child contains much more phenylalanine than a child with PKU can process. Thus, treatment consists of a diet containing just enough phenylalanine as is necessary for the child's growth and development.

Meat, fish, cheese, eggs, nuts, milk, and soy are rich in protein and therefore cannot be used in the diet. Other foods containing lesser amounts of protein, such as potatoes and grains, are given in amounts that keep the phenylalanine level, which must be regularly checked, within safe limits. The intake of these foods should be evenly distributed throughout the day. The permitted amount of each food may change over time even for the same child.

Most fruits, almost all vegetables including leafy greens, sugar, jams, syrups, and fats such as butter, lard, and vegetable oil can be used relatively freely. In addition, there are specially developed and industrially manufactured foods for children with PKU that are low in protein. These include special bread, pasta, cookies and wafers, rice, milk, and other products that can be used quite freely to diversify the baby's diet. You can prepare them in the usual way (frying, boiling, baking, etc.), adding herbs and spices to taste.

High-protein foods such as meat, fish, cheese, and eggs that your child cannot eat are replaced with a special amino acid formula that lacks phenylalanine. These protein substitutes are consumed as drinks or pastes, and in older children, they may be in the form of tablets. These formulas are dispensed by prescription and provide the child with "safe" protein for growth and development. The medical geneticist will explain in detail how to properly prepare and take such a formula.

Protein substitutes are a critical part of the PKU diet. They are given in several doses throughout the day. This helps keep blood phenylalanine levels within safe ranges.

Minerals and vitamins are also important in the child's diet. If they are not already included in the protein substitute, your doctor will prescribe the necessary amounts of these substances as dietary supplements. Give your child only those medications prescribed by the doctor!

In which forms of PKU is pharmacotherapy possible?

For certain forms of PKU, pharmacotherapy has been developed using sapropterin** (sapropterin dihydrochloride**), which is a synthetic analogue of natural tetrahydrobiopterin. The efficacy of the medicinal product is determined by testing.

Testing is performed, monitored, and evaluated by the physician who treats and follows up patients with PKU. For more careful monitoring, it is advisable to perform the test in a hospital setting, possibly a day hospital, or on an outpatient basis provided the patient strictly follows the doctor's testing recommendations.

Before testing, it is very important to stabilize blood phenylalanine levels by maintaining an unchanged diet for 4-5 days before the test begins and during the testing period to eliminate the effect of diet on fluctuations in blood Phe concentration. After obtaining a positive test result, it must be confirmed during a longer follow-up.

Increased tolerance to dietary PA during treatment with synthetic BH4 analogues is an indication for expanding the diet, provided that the PA concentration does not exceed 6 mg/dL. When combining a low-phenylalanine diet with a synthetic BH4 analogue, regular monitoring of the chemical composition of the diet, anthropometric and biochemical parameters of the patient's nutritional status, and blood PA levels is required.

Patients receiving a synthetic BH4 _analogue require careful monitoring of their nutritional status and other health indicators.

To make treatment effective, parents, family members of patients with PKU, and the patients themselves must maintain close contact with their attending physician and follow all medical prescriptions!

How will this affect other children in our family?

Given the autosomal recessive inheritance pattern, the mother and father of an affected child are carriers of the disease, although they are completely healthy. With each pregnancy, the risk of having an affected child is 1 in 4, which is a fairly high risk of recurrence in the family. Therefore, genetic counseling is indicated to help the family have a healthy child. Genetic counseling is also necessary for all patients with PKU of reproductive age.

Adult patients with PKU (regardless of gender) should know that if their partner does not carry mutations in the PAH gene, all children in this marriage will only be carriers, and the disease will not affect their health. On the other hand, if a partner of a patient with PKU carries a mutation in the PAH gene, 50% of their children may suffer from phenylketonuria. If both parents are patients with PKU, all of their children will also have this disorder and will need to receive the same treatment as their parents. Women of reproductive age with a previously established diagnosis of PKU are advised to plan pregnancy in advance, consult a gynecologist and geneticist beforehand, and strictly follow their recommendations. These measures are necessary to reduce the risk of women with PKU giving birth to infants with various congenital malformations ("maternal PKU syndrome").

Guidelines for expectant mothers diagnosed with phenylketonuria and parents of girls with phenylketonuria.

This is important to know!

It is important to remember that after completing specialized treatment, high levels of phenylalanine and its derivatives remain in your blood. This does not have a harmful effect on your body. However, if you are planning to become a mother, high blood phenylalanine levels will become a "severe threat" to your future child. An elevated blood phenylalanine level in a pregnant woman with PKU causes severe fetal malformations of the cardiovascular system and brain, physical and intellectual developmental delay, and epilepsy. A high concentration of phenylalanine and its derivatives in the blood during pregnancy is "toxic" to the developing baby's body.

What needs to be done to prevent the birth of an affected child:

1) Visit a medical genetic consultation center when planning a pregnancy. If DNA diagnostics have not been performed, you and your partner should undergo DNA testing.

2) Register at the medical genetic consultation center and start treatment 2–3 months before the planned pregnancy. During this period, switch to a strict low-phenylalanine diet using a specialized medical nutrition product (amino acid mixture with a low PA content). The blood phenylalanine level at this stage should not exceed 360 µmol/L (6 mg/dL).

3) After conception, blood phenylalanine levels should remain within 120–240 µmol/L (2–4 mg/dL). Up to 12 weeks of pregnancy, blood phenylalanine monitoring should be performed at least once a week. After 12 weeks, at least once every 2 weeks. The medical geneticist will calculate the optimal specialized diet and dosage of the specialized medical nutrition product.

Only compliance with the above actions, ongoing monitoring by a medical geneticist, and scheduled blood phenylalanine level control will help avoid serious complications in your child's development.

Frequency of examinations in patients with HPA

Initial diagnostics

Follow-up of children with HPA

Long-term monitoring of adult patients with hyperphenylalaninemia

Appendix G. Assessment Scales, Questionnaires, and Other Evaluation Tools

Appendix G1. Clinical classification of HFA based on blood Phe levels before treatment

Russian title: Clinical classification of HPA based on pre-treatment blood Phe levels

Original title:

Source: Blau N., Burton B.K., Thöny B. et al. Phenylketonuria and BH₄ Deficiencies// 1st edition-Bremen:UNI-MED. 2010. 94P

Type: rating scale

Purpose: determination of the disease form based on the Phe level

Contents:

Note: **— conversion factor from µmol/L to mg/dL is 60 [33].

Key: contained within the scale itself