Phenylketonuria

　　Phenylketonuria is an autosomal recessive genetic disorder, with the mutated gene located on the long arm of chromosome 12 (12q24.1). Even a slight variation in this gene can cause the disease, not due to gene deletion, but due to the marriage of two heterozygotes, resulting in a genetic disease. It is more common in offspring of consanguineous marriages. About 40% of the siblings of the affected children are also affected. Due to the mutation of the phenylalanine hydroxylase (phenylalanine hydroxylase) gene, there is a deficiency of phenylalanine hydroxylase in the liver, which is the basic biochemical abnormality of the disease. If the variant bases are different, the severity of clinical manifestations can vary greatly, ranging from typical PKU to mild hyperphenylalaninemia.

　　Phenylalanine (phenylalanine, PA) is an essential amino acid for the human body, which participates in the formation of various protein components, but cannot be synthesized in the body. Normally, about 50% of the ingested PA is used to synthesize various protein components, and the rest is converted into tyrosine under the action of phenylalanine hydroxylase (PAH), and then converted into dopamine, norepinephrine, epinephrine, and melanin by other enzymes. Phenylalanine hydroxylase is a complex enzyme system, which includes not only the hydroxylase itself but also dihydropteridine reductase and coenzyme tetrahydrobiopterin. Any enzyme defect can cause an increase in blood phenylalanine levels. When PA hydroxylase is deficient, phenylalanine that does not participate in the first step of protein synthesis accumulates in the plasma and deposits in various tissues including the brain. When the blood phenylalanine exceeds the renal threshold, it is excreted, resulting in phenylalanine aminoaciduria. After the main pathway (hydroxylation) of PA is blocked, the secondary metabolic pathway of PA is compensatorily enhanced, causing PA to be converted into phenylpyruvate, phenyl lactate, normal hydroxyphenylacetic acid, and phenylacetic acid, and the proportion of these metabolites gradually increases. Normally, this metabolic bypass is very little, so the content of these metabolites is very low; when PA hydroxylase is deficient, these metabolites reach an abnormally high level, accumulate in tissues, plasma, and cerebrospinal fluid, and are excreted in large quantities in the urine, resulting in phenylketonuria.

　　As age increases, the amount of phenylalanine consumed for protein synthesis gradually decreases. After birth, the daily intake of phenylalanine is about 0.5g, which increases to 4g in children and adults, of which a large part is oxidized into tyrosine, a process mainly dependent on phenylalanine hydroxylase (PAH), but also requiring cofactors. If this oxidation process is impaired, phenylalanine accumulates in the body, and in this case, phenylalanine is metabolized through other pathways to produce harmful substances such as phenylpyruvic acid. Phenylketonuria (PKU) is a genetic disease caused by reduced or absent PAH activity, and reduced PAH activity can also suppress tyrosine synthesis, leading to reduced melanin production, and inhibit hydroxyphenylpyruvate hydrolase, causing the accumulation of hydroxyphenylpyruvate in the body.

　　Patients may have neurological symptoms and certain physical characteristics, such as rhythmic rocking movements, tremors, active tendon reflexes, and increased muscle tone. Severe patients may have cerebral palsy, and some patients may have epilepsy during infancy, which is often manifested as infantile spasms. The form of epilepsy发作 can change with age.

　　Intellectual development delay, with particularly severe language development disorders. About 80% have abnormal electroencephalograms, which may manifest as high-frequency rhythm disturbances, focal spikes, and so on. The vast majority of children have mental and behavioral abnormalities such as depression, hyperactivity, and autism tendencies, and if not treated promptly and reasonably, they will ultimately cause moderate to severe intellectual disability.

　　Some patients may have skin signs such as eczema. In addition, about 2/3 of the children have mild cranial deformities, normal fundus, no organ enlargement or skeletal abnormalities.

　　Phenylketonuria is a genetic disease, so newborns have hyperphenylalaninemia. Because they have not eaten, the concentration of phenylalanine and its harmful metabolic products in the blood is not high, so most PKU patients appear normal at birth with no clinical manifestations. If newborns are not screened for phenylketonuria, as the feeding time extends, the concentration of phenylalanine and its metabolites in the blood gradually increases, and the clinical symptoms gradually appear. Symptoms usually begin to appear at 3-6 months. At 1 year old, symptoms are obvious. Untreated children gradually show intellectual and motor developmental delays, hair turning yellow, skin becoming pale, the whole body and urine having a special smell of mouse, and often having eczema. As the age grows, the intellectual disability of the children becomes more and more obvious, and about 60% of older children have severe intellectual disabilities. Two-thirds of the children have mild cranial deformities, normal fundus, no organ enlargement or skeletal abnormalities. About 1/4 of the children have epilepsy seizures, which usually appear before 18 months and can be表现为 infantile spasms, nodding seizures, or other forms. About 80% of the children have abnormal electroencephalograms, with the main abnormality being seizures, and a few being abnormal background activity. After treatment, the concentration of phenylalanine in the blood decreases, and the electroencephalogram also improves significantly.

　　We divide the main clinical manifestations of phenylketonuria into three parts: the first is the physical characteristics. (1) The skin is often dry, pale, and delicate, prone to eczema and skin scratch syndrome. Due to the inhibition of tyrosinase, the synthesis of melanin is reduced, so the hair of patients is light-colored, brown, dry, and without luster. The head circumference is small, the eruption of milk teeth is slow, the teeth are sparse, the development of bones is delayed, and the iris color is light. (2) Sweat and urine excrete a bad smell of mouse and moldy smell. (3) Early symptoms include vomiting, irritability, and easy to be irritable. The second is the growth and development characteristics. From 4 to 9 months after birth, there is a significant delay in intellectual development, especially in language development, which suggests a brain development disorder. Some patients have epilepsy seizures, among which some infants have convulsions, which usually appear before 18 months of age. The vast majority of children have mental and behavioral abnormalities such as depression, hyperactivity, and autism tendencies, and if not treated in a timely and reasonable manner, they will eventually cause moderate to severe intellectual disability. The third is the symptoms of the nervous system. The signs are not common, and there may be small brain malformations, increased muscle tension, abnormal gait, recurrent seizures, hyperreflexia, hand tremors, and repetitive limb movements. There are often excitement, restlessness, and abnormal behavior. Early symptoms include vomiting, irritability, and easy to be irritable. Older children may have small seizures and grand mal seizures. The vast majority of children have mental and behavioral abnormalities such as depression, hyperactivity, and autism tendencies, and if not treated in a timely and reasonable manner, they will eventually cause moderate to severe intellectual disability.

　　Phenylketonuria is preventable. The prevention of PKU includes two aspects: the prevention of the onset of PKU and the prevention of the birth of children with PKU.

　　Preventing the onset of PKU requires early diagnosis and early treatment to avoid damage to the nervous system caused by the high metabolism of phenylalanine, phenylacetic acid, and phenyllactic acid. Since it takes time for the accumulation of phenylalanine, phenylacetic acid, and phenyllactic acid in the body to reach the concentration needed to damage the brain, even PKU patients often only have an increase in the concentration of these abnormal metabolic products in the first 1-2 months after birth, which is not enough to cause irreversible damage. If timely diagnosis and effective treatment are provided during this stage, nervous system damage can be avoided. The method of detecting the disease within one month after the baby is born, before the onset of the disease, is called neonatal disease screening, which is an effective method of early diagnosis. Blood is collected from the newborn 3 days after birth to detect the phenylalanine concentration. If the phenylalanine level is elevated, further diagnostic tests are needed. After confirmation, effective treatment is provided, and the phenylalanine and its abnormal metabolites in the blood are reduced to normal levels, achieving the goal of preventing the onset of the disease.

　　The prevention of the birth of children with phenylketonuria (PKU) involves prenatal diagnosis during the fetal stage before birth. If PKU is diagnosed, the parents decide whether to retain the affected fetus. This method of diagnosis before birth is called prenatal diagnosis. Prenatal diagnosis of PKU is suitable for parents who already have a PKU patient and wish to have another child. The method is: first, detect the mutation sites of the pathogenic gene in the blood cells of the child and the parents, which is the premise of prenatal diagnosis of PKU. Then, collect amniotic fluid at 16-20 weeks of the mother's second pregnancy and detect whether the fetus's cells in the amniotic fluid carry two pathogenic gene mutation sites. If they carry two mutation sites, the fetus is a PKU patient. If they carry only one mutation site, the individual is a carrier. This allows for prenatal diagnosis, enabling parents to decide whether to keep the fetus, and can prevent the birth of children with PKU.

　　The treatment of genetic diseases is difficult and the efficacy is not satisfactory, making prevention even more important. This includes avoiding marriage between close relatives, promoting genetic counseling, carrier gene detection, prenatal diagnosis, and selective induced abortion to prevent the birth of affected children; women with PKU should also adopt dietary treatment before and during pregnancy to reduce the blood phenylalanine level to the permissible level of 0.6 mmol/L, otherwise it will bring serious consequences to the fetus. Generally speaking, the phenylalanine level in the mother's blood that is safe for the fetus is between 0.24 and 0.36 mmol/L (4-6 mg). If the hyperphenylalaninemia of a pregnant mother with PKU is controlled, the aforementioned adverse effects on the fetus can be completely avoided. Promoting breastfeeding, early detection of phenylketonuria carriers, and popularizing ferric chloride urine diapers can help identify and treat affected infants early, which is an important method for preventing intellectual disability.

　　Phenylketonuria has the following examination methods:

　　Neonatal screening. After feeding the newborn milk for 3 days, peripheral blood is collected using thick filter paper, dried, and then sent to the screening laboratory. The phenylalanine concentration can be semi-quantitatively measured using the Guthrie bacterial growth inhibition test; it can also be measured by colorimetric quantitation under the action of phenylalanine dehydrogenase, with a lower rate of false negatives. When the phenylalanine content is greater than 0.24 mmol/L (4 mg/dl), which is twice the normal reference value, a re-examination or a venous blood quantitative determination of phenylalanine and tyrosine should be performed. Usually, the plasma phenylalanine level in the child can be above 1.2 mmol/L (20 mg/dl). This is the earliest, most economical, and practical semi-quantitative method for measuring blood phenylalanine. Currently, cities such as Shanghai and Beijing have used this method for neonatal screening to diagnose phenylketonuria patients early.

　　Phenylalanine measurement in blood. The concentration of phenylalanine in normal blood ranges from 60 to 180 μmol/L, while in PKU patients it can be as high as 600 to 3600 μmol/L. This method is used to define a threshold to measure the concentration of phenylalanine in the patient's blood. (1) A threshold of 258 μmol/L separates normal individuals from phenylketonuria patients, with up to 4% false positives. Chromatography can show false negatives in newborns a few days after birth. The MS/MS (mass spectrometry) method can reduce the rate of false positives and can simultaneously measure blood phenylalanine and tyrosine, and calculate the phenylalanine/tyrosine ratio. (2) If the ratio of 2.5 is used as the threshold between normal children and patients, the rate of false positives can be reduced to 1%. Therefore, this method is currently used to screen for neonatal PKU. This method can also be used to screen for galactosemia, maple syrup urine disease, homocystinuria, and congenital hypothyroidism, allowing for the screening of multiple congenital diseases in a single test.

　　Urine examination. (1) Urine ferric chloride test and domestic phenylketonuria rapid diagnostic strip test: If phenylpyruvate is present in the urine, the ferric chloride test will turn green and fade after being placed; if homogentisic acid, histidine, or chlorpromazine metabolites are present in the urine, they may also turn green and show a false positive reaction. If the urine turns blue-green after wetting the domestic PKU test strip with urine, it is positive, and the color depth is compared with the standard color chart to estimate the content of phenylpyruvate in the urine. Since the serum phenylalanine concentration is below 908-1210.6/μmol/L, phenylpyruvate may not be excreted in the urine, so the ferric chloride and test strip tests in the neonatal period may be negative. (2) 2,4-nitrophenylhydrazine test: A yellow precipitate will form when urine is added, indicating a positive result. It should be noted that both are chemical chromogenic methods for detecting phenylpyruvate in urine. Due to their poor specificity, there is a possibility of false positives and false negatives, and they are generally used as an initial screening for older children.

　　Fluorescence spectrophotometry: This method can be used to quantitatively determine phenylalanine.

　　Amino acid chromatography: It is a simpler method for quantifying phenylalanine using finger or heel blood.

　　Amino acid analysis: It is a quantitative method of blood amino acid automatic analysis using an amino acid analyzer. It can differentiate amino acid metabolism diseases by quantifying phenylalanine, tyrosine, and other amino acids, as well as the ratio of branched-chain and aromatic amino acids. Amino acid analysis has an important significance in distinguishing the types of phenylketonuria and in differentiating hyperphenylalaninemia.

　　Phenylalanine tolerance test: After oral administration of 100mg/kg of phenylalanine, the blood phenylalanine level is checked 1-4 hours later. If the content increases and the tyrosine content decreases, it can be diagnosed.

　　Electroencephalogram (EEG) examination: The main findings are spike-wave complexes, and occasional high-amplitude rhythmic disorders. With the increase of age, abnormal EEG manifestations gradually increase, and the abnormal EEG gradually decreases after the age of 12.

　　X-ray examination: Small head畸形 can be seen during pregnancy.

　　CT and MRI examinations: They can detect non-specific changes such as diffuse cortical atrophy.

　　Urine purine analysis: The content of new xanthine and biopterin in urine can be determined by high-performance liquid chromatography (HPLC), which can differentiate various types of PKU: the total excretion of xanthine in the urine of children with PAH deficiency increases, while the ratio of new xanthine to biopterin is normal; children with DHPR deficiency show increased total excretion of xanthine and decreased tetrahydrobiopterin; children with 6-PTS deficiency show increased ratio of new xanthine to biopterin and increased excretion of new xanthine; children with GTPCH deficiency show decreased total excretion of xanthine.

　　DNA analysis: Currently, DNA analysis methods can be used for genetic diagnosis of PAH and DHPR defects. However, due to the numerous polymorphisms of genes, the analysis results must be cautious.

　　It should be noted that this disease is one of the few treatable genetic metabolic diseases, and early diagnosis and treatment should be sought to avoid irreversible damage to the nervous system. Since symptoms do not appear in the early stages, laboratory testing is necessary.

　　Infants can be fed with specially formulated low phenylalanine milk powder. When adding complementary foods for young children, low-protein foods such as starches, vegetables, and fruits should be the main focus. Since phenylalanine is an essential amino acid for protein synthesis, its deficiency can also lead to nervous system damage, so it is still appropriate to supply it in an amount of 30-50mg/kg per day to maintain the blood phenylalanine concentration at 0.12-0.6mmol/L (2-10mg/dl).

　　The treatment of phenylketonuria requires specific treatment measures according to the type.

　　The key to treating typical PKU is to control the content of phenylalanine (PA) in the diet and adopt a low-phenylalanine diet. During infancy, artificial low-phenylalanine formula can be used for feeding, which is also produced in China. It can meet the minimum needs of the body's metabolism and growth and development without causing brain damage due to excessively high PA levels in the blood. The development of the human brain is most important in the first year after birth (especially the first six months), so dietary treatment should start from newborns. If treatment begins after 6 months of age, the efficacy is poor; starting at 4-5 years old is ineffective because the development of the nervous system is basically complete (Holtzman et al., 1986). If the PA content in the child's diet is controlled, maintaining the blood PA level at 0.18-0.92 mmol/L (5-10 mg/dl) may achieve ideal efficacy, which can improve intelligence development. However, maintaining a low-phenylalanine diet after weaning is not easy, as the PA content in both animal and plant proteins can reach 0.18-0.31 mmol/L. It is necessary to strictly limit protein intake and fully meet the needs of calories, fats, vitamins, and minerals, which requires a nutritionist to plan the diet and conduct strict follow-up. Special diets have a poor taste, and many children find it difficult to accept them. Literature reports that better dietary control leads to higher IQ development. If dietary treatment is interrupted during childhood, mental and motor developmental delays and a decrease in IQ may occur; if dietary treatment is resumed, it is still possible to achieve some efficacy. Many cases in China are diagnosed when brain damage has already occurred in the late infancy or childhood, at which time dietary treatment cannot improve intelligence quotient, but it is still beneficial for controlling seizures, improving developmental delays, eczema, hypopigmentation of the skin and hair, scleroderma, reduced seminal fluid, and reproductive dysfunction. Currently, there is still controversy over the duration of dietary treatment. Most children do not need to strictly limit their diet after the age of 6, but the intake of phenylalanine is still limited. Children with phenylalaninemia but without PKU do not need dietary treatment. Some scholars also believe that dietary treatment should continue until after adolescence and even throughout life. Adequate consideration must be given to the side effects of dietary treatment, such as growth retardation, short stature, low weight, hypoglycemia, hypoalbuminemia, and delayed bone age. Since PA is an essential amino acid for the human body, complete deficiency can lead to serious consequences such as drowsiness, anemia, anorexia, diarrhea, and rash, and even death. It should be noted that during the dietary treatment process to limit phenylalanine intake, the child's growth and development, nutritional status, blood phenylalanine levels, and side effects should be closely observed. The main side effects are other nutritional deficiencies, which can lead to diarrhea, anemia (macrocytic), hypoglycemia, hypoalbuminemia, and pellagra-like rash.

　　A few children with PKU variants cannot prevent nervous system involvement by restricting PA diet. Some of these children have dystonic extrapyramidal muscle rigidity as early as neonatal early stage, known as stiff-baby syndrome. Biopterin (biological biopterin) may be effective because the level of phenylalanine hydroxylase in their liver is normal. The deficiency of this enzyme is due to the synthesis disorder of the tetrahydrobiopterin activator, which can be caused by the deficiency of dihydrobiopterin degradation enzyme or the reduction of biopterin (biological biopterin) synthesis. The levels of catecholamine metabolites and 5-hydroxytryptamine in urine are reduced, and dietary PA restriction is ineffective. The treatment for these cases is mainly corrected by supplementing neurotransmitter precursors levodopa (L-dopa) and 5-hydroxytryptophan (5-hydroxytryptophan).