Phenylketonuria

　　First, etiology

　　This disease is an autosomal recessive genetic disease caused by the defect of enzymes in the phenylalanine metabolic pathway.

　　Second, pathogenesis

　　1, Phenylalanine (phenylalanine, Phe) is an essential amino acid for the human body. Part of the Phe ingested into the body is used for protein synthesis, and part is converted into tyrosine through the action of phenylalanine hydroxylase. Only a small amount of Phe is converted into phenylpyruvate through secondary metabolic pathways under the action of transaminases.

　　2, PKU is caused by gene mutation of phenylalanine hydroxylase (phenylalanine hydroxylase, PAH), leading to reduced or lost activity of PAH, and metabolic disorder of Phe in the liver. PKU patients lack phenylalanine hydroxylase, resulting in reduced tyrosine and normal metabolic products, increased blood Phe content, stimulating the development of transaminases, enhancement of secondary metabolic pathways, and the formation of phenylpyruvate, phenylacetic acid, and phenyllactic acid, which are excreted in large quantities in urine, hence the name phenylketonuria. Phenyllactic acid makes the urine of children have a special smell of mouse urine. High concentrations of Phe and its abnormal metabolites inhibit tyrosinase, causing障碍 in melanin synthesis. Increased Phe affects brain development, leading to delayed intelligence development and the appearance of symptoms such as microcephaly, convulsions, and spasms in the nervous system.

　　3, The PAH gene of humans is located on chromosome 12 (12q22～12q24.1), with a full length of about 90kb. It has 13 exons and 12 introns, with exon lengths ranging from 57 to 892bp, and mature mRNA is about 2.4kb, encoding 451 amino acids. The length of introns is between 1 and 23kb. With the development of molecular biology technology, Beijing, Shanghai and other places have carried out gene analysis on PKU patients using techniques such as single-strand conformation polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), dot hybridization, and DNA sequence analysis. More than 30 kinds of gene mutations have been found in the Chinese population, and the mutations of exons 7 and 12 account for a relatively high proportion. Among them, there are some mutations specific to Chinese people, which cause amino acid substitution, premature termination of translation, abnormal mPNA splicing, and frame shift in reading frame.

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

　　2, Intellectual development delay, especially severe language development disorders. About 80% have abnormal electroencephalogram, which may manifest as peak rhythm disorder, focal spike waves, etc. 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 impairment.

　　3. Some patients may have skin signs such as eczema.

　　According to different clinical types, PKU can be divided into:

　　1. Classical PKU (classical PKU):The patients have typical clinical manifestations, varying degrees of intellectual disability, 60% belong to severe disability (IQ below 50), about 1/4 of the patients have seizures, the patients have light hair and skin color, urine and sweat emit a mouse odor, accompanied by mental and behavioral abnormalities, blood Phe concentration > 1200μmol/L (20mg/dl), urine FeCl3-and DNPH test is strongly positive.

　　2. Moderate type PKU (moderate PKU):Clinical symptoms are relatively mild, laboratory examination results are the same as classic PKU, but blood phenylalanine levels are between 360-1200μmol/L, children respond well to treatment, and blood phenylalanine concentration is easier to control than in classic PKU patients.

　　3. Mild PKU (mild PKU):Clinical symptoms are mild or asymptomatic, blood phenylalanine levels are less than 120-360μmol/L, seen in a very few newborns or premature infants, or in those with high residual enzyme activity of phenylalanine hydroxylase.

　　4. Tetrahydrobiopterin (BH4):Clinically, all blood phenylalanine levels greater than 120μmol/L are called hyperphenylalaninemia. From the etiology, hyperphenylalaninemia is divided into two major categories: phenylalanine hydroxylase deficiency and the cofactor of PAH - tetrahydrobiopterin (tetrahydrobiopterin, BH4) deficiency. The treatment methods for the two types of hyperphenylalaninemia are different, and early differential diagnosis is very important.

　　How to prevent phenylketonuria in children:

　　Avoid consanguineous marriage, and there should be no marriage between heterozygotes. Newborn screening should be carried out to detect PKU patients early, start treatment early to prevent intellectual disability. For high-risk families, prenatal diagnosis can be performed to decide whether to have selective artificial abortion. Couples with a family history of the disease must use DNA analysis or methods such as amniotic fluid purine to perform prenatal diagnosis on their fetus.

　　1. Neonatal screening

　　1, Patients with phenylketonuria (PKU) in the neonatal period show no clinical symptoms, and symptoms of PKU gradually appear after 3 months of birth. With the development of preventive medicine science, neonatal screening for phenylketonuria has gradually become routine. Neonatal screening is to determine blood phenylalanine and screen each newborn in the population, so that PKU patients can be diagnosed early before the onset of clinical symptoms, and early treatment can be given to avoid intellectual disability. Urine ferric chloride (FeCl3) and 2,4-dinitrophenylhydrazine test (DNPH)

　　2. These two tests may also be positive in maple syrup urine disease, cystinuria, so they are not specific tests for PKU, and further blood phenylalanine determination is needed for diagnosis. Neonatal PKU, because the metabolic bypass of phenylalanine metabolism has not been fully developed, the urine of patients is tested negative, and this method cannot be used for neonatal screening.

　　II. There are two methods for blood phenylalanine determination

　　1. Guthrie bacterial inhibition method: the normal concentration is 1200μmol/L.

　　2. Phenylalanine fluorescence quantification method: the normal value is the same as the bacterial inhibition method.

　　III. Phenylalanine loading test

　　For blood phenylalanine concentration greater than the normal concentration, 1200μmol/L is diagnosed as PKU,

　　IV. HPLC urine xanthine analysis

　　0ml morning urine is added with 0.2g vitamin C, acidify the urine after that, make the 8cm×10cm neonatal screening filter paper moistened, dried, sent to the laboratory with conditions for analysis of urine xanthine, and carry out the diagnosis and differential diagnosis of tetrahydrobiopterin deficiency.

　　V. Oral tetrahydrobiopterin loading test

　　In the case of blood Phe concentration > 600μmol/L, directly give oral BH4 tablets at a dose of 20mg/kg. Before and 2, 4, 6, 8, 24 hours after taking BH4, blood is taken for Phe measurement, for blood Phe concentration

　　1. Edible oils include lard, beef fat, butter, vegetable oil, sesame paste, etc. Other foods such as honey, sucrose, candy, etc., can also be used.

　　2. Provide sufficient fat and carbohydrates to meet the body's energy needs. You can choose wheat starch, cornstarch, lotus root starch, potato starch, substitute lotus root starch, noodles, noodles, jelly, pumpkin, lotus root, carrot, yam, etc. Because these foods contain low phenylalanine and high carbohydrates, they can satisfy hunger.

　　3. During the breastfeeding period, low-phenylalanine hydrolyzed protein can be used to replace human milk or milk. If milk is used, it should not exceed 250 grams per day, and other additives such as lotus root powder, vegetable water, and vegetable puree can be added.

　　4. Eat more fresh vegetables and fruits to supplement vitamins. Such as eggplant, onion, persimmon, cabbage, round cabbage, rapeseed, etc.

　　5. Phenylketonuria is directly related to the dietary content of phenylalanine, and low-phenylalanine dietary treatment should be started as soon as possible. The content of phenylalanine in the diet is calculated at 15-30 mg per kilogram of body weight per day, and the total protein content is 2-4 grams per kilogram of body weight per day. Since any food protein contains 4-6% phenylalanine, all protein-rich foods such as milk, eggs, lean meat, dried beans, and bean products must be avoided or used sparingly.

　　1. Precautions before the treatment of pediatric phenylketonuria

　　1. Prevention: Avoid consanguineous marriage. Mate should not be matched between heterozygotes. Carry out neonatal screening to early detect children with PKU, start treatment early to prevent intellectual disability. Every newborn should undergo a diaper test to facilitate early diagnosis and treatment.

　　2. Couples with a family history of the disease must use DNA analysis or tests for hypoxanthine in amniotic fluid to perform prenatal diagnosis on their fetus. This will determine whether to perform selective induced abortion.

　　3. Currently, about 80% of the gene mutations in Chinese PKU patients have been identified, and about 20% of the mechanisms of gene mutations are unknown. Each PKU family has two mutation genes, so gene diagnosis can have three results:

　　4. Both mutation genes can be diagnosed clearly; 2. One mutation gene can be diagnosed clearly, while the other cannot; 3. Both mutation genes cannot be diagnosed clearly. The first two results can provide prenatal diagnosis. Under the premise of distinguishing non-classical PKU, indirect genetic diagnosis can also be performed through linkage analysis, and prenatal diagnosis can also be carried out.

　　5. Since PKU is a genetic disease, blood samples from the child and parents are required for genetic diagnosis. Moreover, due to the variety of mutation genes and the complexity of analysis, genetic diagnosis should be conducted about six months to a year before the next pregnancy. This allows for targeted prenatal diagnosis based on the results of genetic diagnosis during pregnancy.

　　6. Phenylketonuria can be detected in blood and urine within a few days after birth. Therefore, many countries have listed this disease as part of neonatal screening.

　　2. Western Treatment Methods for Paediatric Phenylketonuria

　　1. Treatment Principles

　　(1) Treatment of complications and relief of symptoms. For phenylketonuria itself, there is no medication available for treatment. Once diagnosed, treatment should be initiated as soon as possible, primarily through dietary therapy. The younger the age at the start of treatment, the better the outcome.

　　(2) PKU is the first hereditary metabolic disease that can be treated through dietary control. Natural foods contain a certain amount of phenylalanine, and a low-protein diet can lead to malnutrition. Therefore, a low-phenylalanine diet should be used for treatment:

　　(3) Early Treatment: Once diagnosed, treatment should be initiated immediately. The younger the age at the start of treatment, the better the prognosis, and the intellectual development can approach that of normal individuals. Patients treated after the age of 3 to 5 may experience a reduction in seizures and behavioral abnormalities, but there is no significant improvement in existing severe intellectual disabilities. With the gradual promotion and popularization of neonatal screening in China, patients identified through screening often receive diagnosis and treatment within one month, and sometimes even two weeks of birth, ensuring the healthy growth of the child.

　　(4) Control of Phenylalanine Intake: Phenylalanine is an essential amino acid, necessary for growth and metabolism within the body. The intellectual disability in PKU patients is caused by the neurotoxic effects of excessive Phe and by-products of the bypass metabolism, leading to brain damage. To prevent brain damage, the intake of phenylalanine from food must be reduced. Blood phenylalanine levels should be controlled within a certain range to meet the needs of growth and development. Ideally, blood phenylalanine concentration should be maintained between 120 and 360 μmol/L. Over-treatment can lead to phenylalanine deficiency, causing drowsiness, anorexia, anemia, diarrhea, and even death.

　　(5) Survey of individual dietary regimens: Since each child has a different tolerance level for phenylalanine, the dietary regimen should still be adjusted according to the specific situation of the child during dietary treatment. Low-phenylalanine formula milk treatment should last at least until the age of 12.

　　(6) Parental cooperation: It is one of the key factors for success. If parents have a full understanding of the treatment principles and the dietary control is relatively reasonable, the intellectual development of the child is often normal.

　　(7) Female patients before pregnancy: Adult female patients should resume dietary control before pregnancy and continue until delivery to avoid hyperphenylalaninemia affecting the fetus.

　　(8) Prenatal diagnosis in high-risk families: In recent years, prenatal diagnosis for PKU high-risk families has been carried out in cities such as Beijing and Shanghai. By directly searching for gene mutation points and combining microsatellite genetic polymorphism analysis methods (STR, VNTR), prenatal diagnosis has been successfully implemented for high-risk families, achieving good social benefits. Before prenatal diagnosis, venous blood samples from PKU children and their parents must be collected for pedigree linkage analysis. Prenatal diagnosis involves collecting chorionic villi at 9-12 weeks of pregnancy or amniotic fluid cells at 16-18 weeks. Since STR polymorphism linkage analysis does not directly detect gene mutations, attention must be paid to the accuracy of clinical diagnosis when applying it, and it should never be used to analyze linkage in cases of PKU with non-PAH gene mutations as if they were PAH mutation cases. In prenatal diagnosis, it is also necessary to strictly prevent sample contamination, especially maternal cell contamination.

　　2. Treatment Methods

　　(1) A low-phenylalanine diet is mainly suitable for typical PKU and patients with blood phenylalanine levels consistently above 1.22 mmol/L (20 mg/dl). Therefore, infants can be fed with specially formulated low-phenylalanine formula milk, and during the weaning period, supplementary foods should mainly include low-protein foods such as starches, vegetables, and fruits. The phenylalanine requirement is approximately 50-70 mg/(kg.d) for the first 2 months, 40 mg/(kg.d) for 3-6 months, and 25-30 mg/(kg.d) for 2 years, and about 10-30 mg/(kg.d) for those over 4 years old, with the aim of maintaining blood phenylalanine concentration between 0.12-0.6 mmol/L (2-10 mg/dl). Dietary control should last at least until after puberty. The addition of tyrosine to the diet can restore the normal coloration of hair but has no effect on intellectual progress. Close observation of the child's growth, development, nutritional status, blood phenylalanine levels, and side effects should be made during the dietary treatment process that restricts phenylalanine intake.

　　(2) Dietary treatment must also take into account individual differences. Due to the great variation in the activity defect of phenylalanine hydroxylase in children, dietary treatment must adhere to the principle of individualization. Moreover, because of the different protein, calorie, phenylalanine requirements and tolerance levels in children of different age groups, dietary regimens should be formulated and adjusted according to each child's age, weight, and blood Phe concentration to control the blood phenylalanine concentration at an appropriate level.

　　3. Drug Treatment:

　　(1) In addition to dietary treatment for atypical phenylketonuria, multiple neurotransmitters should be supplemented, such as BH4, dopamine, 5-hydroxytryptamine, folic acid, etc. Symptomatic treatment should be given to children with other complications. For example, children with epilepsy should start regular antiepileptic drug treatment as soon as possible. Children with eczema can recover spontaneously after satisfactory control of the blood Phe concentration. If eczema is severe, symptomatic treatment with topical medication can be given. Intellectual backwardness caused by brain damage is irreversible, but there can be varying degrees of improvement after intellectual rehabilitation, and some may even have significant progress. For families that meet the conditions, consider training for intellectual rehabilitation for the children. For children with severe intellectual retardation, the purpose of training is to cultivate basic self-care abilities, and for children with mild to moderate intellectual retardation, in addition to cultivating their living skills, appropriate survival skills training should also be carried out.

　　(2) Biopterin (biopterin, BH4) is used to treat variants that do not respond to a low phenylalanine diet. Initially, the dose of biopterin (BH4) used was 2.5mg/kg per day. Since biopterin (BH4) penetrates the blood-brain barrier poorly, most children still have to be combined with a low phenylalanine diet and supplemented with levodopa 10-15mg/kg per day, 5-hydroxytryptamine 4mg/kg per day, and carbidopa (Carbidopa) (a decarboxylase inhibitor) 1-2mg/kg per day. Kapatos et al. proved that with high-dose biopterin (BH4), effective therapeutic concentrations can be reached in the brain tissue, and in view of the adverse effects such as hallucinations, motor disorders, and psychiatric symptoms that can be caused by long-term use of levodopa, 5-hydroxytryptamine can also cause scleroderma-like lesions. Therefore, an attempt was made to treat this type of PKU with high-dose BH4 alone, with the maximum dose reaching 40mg/kg per day. Since each child's sensitivity to BH4 is different, the treatment dose for each case should be adjusted according to the following clinical indicators.