Introduction to hyperphenylalaninemia

Hyperphenylalaninemia(hyperphenylalaninemia, HPA) is due to phenylalanine hydroxylase (PAH) deficiency or its coenzymeTetrahydrobiopterin (BH4) deficiencyA group of the most common amino acid metabolic diseases that cause an increase in phenylalanine (Phe). With the development of disease diagnosis, differential diagnosis, treatment, neonatal screening and prevention technology, HPA has become a treatable and preventable disease, and is a model for the history of prevention and treatment of genetic metabolic diseases [1-3].

China began HPA screening and treatment research in 1981. In the past 33 years, the national screening rate has been gradually improved [4-6]. The diagnosis of most HPA children from the diagnosis of clinical symptoms is to the asymptomatic biochemistry and genes of newborns. Diagnostic changes, early screening, differential diagnosis, and proper treatment of HPA are becoming new challenges for clinicians.

To this end, according to the Ministry of Health "Phenylketonuria and congenital hypothyroidism diagnosis and treatment of technical specifications" [7], with reference to domestic and foreign experience and guidelines [8-10], Chinese Medical Association Pediatric Branch Endocrine Genetic Metabolism Group and The Newborn Screening Group of the Chinese Society of Preventive Medicine's Birth Defect Prevention and Control Committee discussed the diagnosis and treatment of HPA and proposed the following consensus.

Overview

1.1 HPADefinition

Blood Phe concentration > 120 μmol / L (> 2 mg / dl) and blood Phe and Tyrosine (Tyr) ratio (Phe / Tyr) > 2.0 are collectively referred to as HPA [8-10].

1.2 HPACause and classification

The causes of HPA are divided into PAH deficiency and BH4 deficiency, both of which are autosomal recessive diseases [1,9,11].

PAH deficiency: Children with varying degrees of HPA due to PAH deficiency (Figure 1). It is usually classified according to the highest blood Phe concentration before treatment or the natural protein intake sufficient for blood Phe concentration. Classical phenylketonuria (PKU): blood Phe ≥ 1 200 μmol / L; mild PKU: blood Phe 360 ~ 1200 μmoI / L; mild HPA: blood Phe 120 ~ 360 μmoI / L [1, 11 ].

Foreign countries also classify dietary Phe tolerance according to 2~5 years old. Children with classic PKU, mild PKU and mild HPA** have a tolerance to Phe <20 20=” 50=”” kg ="" > 50 mg / (kg · d) [1,8], but the need for standardized post-diet assessment, clinical practical application is complex.

In addition, the therapeutic response to BH4 based on blood Phe concentration can be divided into BH4 reactivity and BH4 non-reactive PAH deficiency (Fig. 1).

BH4 deficiency: BH4 deficiency is a disorder of HPA and neurotransmitter synthesis caused by a deficiency in one of the five enzymes in the BH4 metabolic pathway. Among them, 6-pyruvoyl tetrahydropterin synthase (PTPS) is the most common, followed by dihydropteridine reductase (DHPR) deficiency, and guanosine triphosphate cyclase (GTP cyclohydrolase). , GTPCH ), sepiapte rinreductase (SR) and pterin-4α-carbinolamine dehydratase (PCD) deficiency are rare (Fig. 1) [3,9,12] . 96% of 256 patients with BH4 deficiency in China were PTPS deficient, and DHPR deficiency accounted for 2.4% [13].

According to cerebrospinal fluid neurotransmitter metabolites or clinical nervous system symptoms, BH4 deficiency is classified into severe type and light type. In severe cases, the neurotransmitter metabolites in cerebrospinal fluid are reduced, and neurological symptoms appear in the clinic. The neurotransmitter metabolites in the cerebrospinal fluid of the mild type are normal and have no neurological symptoms [12, 14, 15].

HPAIncidence and disease spectrum

The incidence and disease spectrum of HPA vary from country to country [1]. China's 1985-2011 screening of 35 million newborns showed that the prevalence rate was 1:10 397 [16-18].

According to international data, PAH deficiency is 98% in Caucasian patients with HPA and about 2% in BH4 deficiency [12,14]. According to China's newborn screening data from 2000 to 2007, 12.9% of HPA is BH4 deficiency, and PTPS deficiency is the most common [13,18], and there are significant regional differences. The incidence of BH4 deficiency in the southern region is higher. [19-21], Taiwan has the highest incidence [22-23].

HPAPathogenesis

Under normal conditions, Phe produced by protein decomposition in natural foods is converted to tyrosine by the action of liver PAH. PAH deficiency can lead to HPA, enhanced bypass metabolism, and a large amount of phenylpyruvate, phenylacetic acid and phenylacetic acid are excreted from the urine. [1,3]. BH4 is a coenzyme of PAH, tyrosine and tryptophan hydroxylase. Any deficiency of BH4 synthesis or reductase can lead to the synthesis of HPA, neurotransmitter dopamine and serotonin. In addition, the increased blood Phe passes through the blood. The brain barrier leads to abnormalities in the brain Phe, causing cerebral myelin dysplasia or demyelination and other abnormal white matter leading to nervous system damage [1,3].

HPAClinical manifestation

There are many clinical symptoms in the neonatal period. After 3 to 4 months of birth, the clinical features of typical PKU are gradually manifested: hair changes from black to yellow, skin color is light, urine, sweat and rat smell, with age, intelligent development is obvious, microcephaly, seizures (more manifestations of seizures), behavior, personality, neurocognition and other abnormalities, such as hyperactivity, self-harm, attack, autism, inferiority, depression, etc. [1,24-25].

Due to the lack of specificity of symptoms, clinical diagnosis is difficult, and it is easy to be misdiagnosed as neurological diseases such as cerebral palsy and epilepsy. It is necessary to rely on biochemical analysis for etiological diagnosis [19,24-25].

In addition to the symptoms of PKU, children with BH4 deficiency are mainly characterized by low trunk muscle tone, increased or decreased muscle tension in the limbs, such as difficulty swallowing, increased saliva, softness, and angulation, etc. [13,15,21].

HPADiagnosis and differential diagnosis

5.1 diagnosis

Newborn screening: The newborn heel blood was collected 72 hours after birth (6-8 times of breastfeeding), and made into a special dry blood filter paper. The blood concentration of Phe was determined by fluorescence or tandem mass spectrometry (MS/MS) for HPA. Screening. Premature infants may cause temporary HPA due to immature liver function. Fever, infection, parenteral nutrition or blood transfusion may also lead to an increase in blood Phe concentration. Insufficient protein intake may lead to false negatives. Conduct a review. Screen the original specimen blood Phe concentration > 120 (μmol / L, or accompanied by Phe / Tyr > 2.0 is positive, call for recovery, the review is still positive, the following differential diagnosis is required (Figure 1) [3,7] .

HPA diagnosis: For newborn screening or high-risk screening of blood Phe, it is recommended to use quantitative method (fluorescence or tandem mass spectrometry) to determine the blood Phe, Tyr concentration, calculate Phe / Tyr, exclude other causes Increased Phe in the blood, such as tyrosinemia, Hitler's protein deficiency, etc. (blood Phe > 120 μmol / L, Phe / Tyr < 2. phe = "" > 120 μmol / L and Phe / Tyr > 2.0 Diagnosed as HPA (Figure 1) [1,3,7].

5.2 Differential diagnosis

For all children with confirmed HPA, the following tests must be performed prior to treatment to identify the cause. For patients with a blood Phe concentration >360 μmol/L, a low-Phe diet may be administered as appropriate after completion of the differential diagnosis test, and the treatment plan may be adjusted according to the differential diagnosis results (Table 1, Figure 1) [1, 3, 7].

Urinary pterin analysis: It is an important method for the diagnosis of BH4 synthase (PTPS, GTPCH) deficiency in China. Immediately after collecting fresh urine, add ascorbic acid (10 to 20 mg of ascorbic acid per 1 ml of urine), mix it in the dark and store it at -20 °C, or infiltrate the ascorbic acid-treated urine into the dry filter paper ( 5 cm x 5 cm), protected from light and dried, sealed and stored in the laboratory [3].

High-performance liquid chromatography was used to determine neopterin (N) and biopterin (B), and the ratio of biopterin B% [B/(B + N) x100%] was calculated. Children with various enzyme deficiencies presented different urinary pterin profiles (Table 1) [3, 9, 12].

Determination of erythrocyte DHPR activity: a diagnostic method for DHPR deficiency. The red blood cell DHPR activity in the dry filter paper blood samples is determined by a dual beam spectrophotometer. DHPR activity was significantly reduced in children with DHPR deficiency (Table 1, Figure 1) [3, 12, 14].

BH4 stress test: A method for the diagnosis of BH4 deficiency and a method for determining BH4 reactive PKU/HPA should be performed after the specimen of urinary pterin is obtained (Fig. 1). For patients with mild HPA or decreased blood Phe concentration after special diet therapy, urine sputum analysis and DHPR activity measurement may be performed first, and BH4 load test may be performed for those with uncertain diagnosis [3, 9, 21]. Normal diet before and during the test. The specific methods and judgments are as follows:

24-hour BH4 stress test: clinical practice suggests that the BH4 load test is a more feasible auxiliary diagnostic method for BH4 deficiency. When the basal blood of newborns is Phe >400 μmol/L, BH4 tablets (20 mg/kg) can be taken orally 30 minutes before feeding (BH4 tablets are dissolved in water), before BH4, 2, 4, 6, 8, 24 after serving. h The blood concentration was measured by Phe concentration, and the urine pheromone spectrum was analyzed after 4-8 hours.

For those with a slight increase in Phe, a normal protein diet is recommended for 3 days. After the blood Phe is increased, the BH4 load test is performed. It is not recommended to do the Phe + BH4 combined load test. Phe + BH4 combined load test is not performed on those with normal basal blood Phe concentration, which may lead to false positives. The blood Phe concentration in children with PTPS deficiency decreased to normal after 2-6 hours after taking BH4, and the blood Phe decreased slowly in DHPR deficiency [12,14,21].

BH4 load test for 2d or longer: For children with normal urinary pterin and DHPR activity, this test helps identify BH4-reactive PKU/HPA. Blau et al [9,12] suggested a 2 h BH4 stress test: blood Phe was measured at 8, 16, and 24 h after oral administration of BH4 (20 mg/kg) for 2 consecutive days. Blood Phe decreased by 8 to 24 h after oral administration of BH4. If it is more than %, it is judged to be BH4 reactive PKU/HPA; the non-responder can be extended for 1 to 2 weeks, and the test still has no reaction, and it is judged that BH4 is unreactive PKU/HPA.

Genetic diagnosis: It is a method for the diagnosis of the cause of HPA. It is recommended to carry out routine diagnosis, especially for those who cannot be clearly diagnosed by the above differential diagnosis test.

*PAH gene: *PAH gene is located on chromosome 12q22-24. 1, full length of about 90 kb, contains 13 exons, encoding 451 amino acids [1,13]. Up to now, nearly 800 PAH gene mutation types (http:// and) have been reported internationally, which are highly genetically heterogeneous and have significant regional and ethnic differences [11, 26-27]. The distribution of PAH gene mutations in children across China is different [27-29].

BH4-related genes: A variety of BH4-deficient-associated gene mutations (.org) have been reported so far. The gene PTS encoding the PTPS enzyme is located at 11 q22. 3-q23. 3, containing 6 exons, and 43 PTS gene mutation types have been found in East Asia.

The Chinese PTS gene hotspot mutation is c. 155A > G, c. 259C > T, c. 286G > A and c. IVS1-291A > G (76.9%), c. 155A > G, c. 259C > T c. 286G > A causes severe PTPS deficiency, c. 166G > A and c. IVS1-291A > G may be associated with mild PTPS deficiency. The DHPR gene is located at 4p15.3 and contains 7 exons, and 35 gene mutation types have been reported [13, 22-23].

5.3 Other auxiliary inspection

Skull imaging studies can help assess the extent of brain damage in children. MRI is superior to CT in assessing the extent of white matter lesions. Untreated or poorly treated children may have brain atrophy and abnormal white matter, myelin dysplasia and/or demyelinating lesions, white matter vacuolar degeneration and angioedema [3, 31-32].

Magnetic resonance spectroscopy (1H-MRS) analysis is a non-invasive technique for detecting the concentration of Phe in the brain of children with PKU, but the technical difficulty is high, and it is difficult to popularize and apply [33]. Patients without early treatment are often accompanied by EEG abnormalities, and EEG should be performed in patients with epilepsy.

HPA treatment

Both PKU and BH4 deficiency are treatable genetic metabolic diseases that require multidisciplinary management, including specialists in genetic metabolic diseases, dietitians, psychology and neurologists, social workers, and government funding.

6.1 PAHDeficiency

Treatment indication: PKU patients with normal P-concentration of blood concentration >360 μmol/L should be treated immediately after the differential diagnosis test is completed. The sooner the better the treatment, the longer-term treatment is advocated; mild HPA can not be treated temporarily, but Blood Phe concentration should be measured regularly. If the blood Phe concentration is continued for 2 times > 360 μmol/L, it should be treated.

Dietary therapy: Low phenylalanine diet therapy is still the main treatment for current PAH deficiency. PKU patients have different PAH enzyme activities, leading to individual differences in Phe tolerance, requiring individualized treatment [8,10]. The treatment method was adjusted according to the daily protein requirement, blood Phe concentration, Phe tolerance, dietary preference, etc. of children in the corresponding age group [8,10].

Neonatal and infancy: feeding is mainly based on dairy diet, and the treatment compliance is better. Children with classic PKU can suspend breast milk or ordinary infant milk powder, and give Phe special milk powder. After 3 ~ 7 days of treatment, the blood Phe concentration drops to normal, and gradually add a small amount of natural milk. The preferred breast milk (Phe content is 1/3 of milk) ), or ordinary baby milk powder or low Phe food supplement [3,8,10]. Mild PKU was prepared according to the blood Phe concentration by 3:1 or 2:1 without Phe special milk powder and ordinary milk powder, and the dietary compatibility was adjusted according to the blood Phe concentration.

Early childhood and childhood: Due to the temptation of natural diet, treatment compliance declines, special milk powder demand decreases, and dietary treatment faces challenges. In order to meet protein needs and blood Phe concentration control, Phe protein powder and/or milk powder can be used to reduce natural protein. According to the individual Phe tolerance, refer to the “Chinese Food Composition Table” to select natural foods with different Phe contents [3, 8, 10].

Foods with high Phe content (such as meat, cheese, fish, eggs, flour, nuts, and soy products) should be avoided in the daily diet; foods with moderate Phe content (including rice, milk, breakfast wheat, potatoes, cream) or Starchy foods, fruits, vegetables, etc. with low Phe content [3, 8, 34].

Adolescents and adulthood: About 75% of adolescents and adult PKU patients have poor treatment compliance. If the treatment is interrupted or the blood Phe is not ideally controlled, it will lead to a series of mental and behavioral abnormalities [1, 35-36]. .

In particular, the increase in blood Phe concentration in pregnant women can lead to fetal brain development disorders and various malformations, namely maternal PKU syndrome [1,37,38]. Therefore, prenatal genetic counseling is required for female patients with PKU. Dietary therapy is required from 6 months before pregnancy to the whole pregnancy, and the blood Phe is controlled at 120-360 μmol/1 [1, 37-38].

BH4 treatment: for children with BH4-responsive PKU, especially those with poor dietary compliance, foreign reports of oral BH4 5 ~ 20 mg / (kg · d), divided into 2 times, or combined with low Phe diet, can improve children with Phe Tolerance, appropriate increase in natural protein intake, improve quality of life and nutritional status [39-41]. Through BH4 load test diagnosis and PAH gene analysis, Chinese counterparts have studied BH4 reactive PKU [42-44], but the application of treatment is limited.

Propaganda and psychological guidance: Parents of newly diagnosed PKU children need to enter the basic knowledge of PKU (including genetic methods, diagnosis and treatment and follow-up principles, etc.) to improve treatment compliance and achieve good results. After entering the school, you need to inform the school teacher to cooperate with the diet and education guidance, and do a good job in psychological counseling for children [1,3].

Other exploratory treatments: Due to the decline in PKU long-term dietary treatment compliance, poor Phe food taste, and special diets that lead to nutritional deficiencies, dietary treatment often faces challenges. Other therapeutic methods have been developed with macromolecular neutral amino acids (LNAA). , Phe-free natural protein glycomacropeptide (GMP) extracted from cheese whey [45-47]. Oral preparation of phenylalanine deaminase, enzyme replacement therapy, gene therapy, etc. are in the experimental stage [48-49].

6.2 BH4Deficiency

Children diagnosed by newborn screening have no clinical symptoms, and it is difficult to judge severe type and light type. After the diagnosis is clear, BH4 or Phe-free diet and neurotransmitter precursor treatment can be given according to different causes, and life-long treatment is advocated [12, 14-15].

BH4 or special diet treatment: the goal is to reduce blood Phe concentration. Patients with PTPS deficiency, GTPCH deficiency and PCD deficiency were supplemented with BH4 [1 ~ 5 mg / (kg · d)] under normal diet, and taken orally twice to control the blood Phe to normal levels. Patients with DHPR deficiency and BH4 treatment difficulties were treated with low Phe special milk powder or diet (treated with PKU) to control blood Phe concentration to near normal levels (120 ~ 240 pmd/L) [12, 14-15].

Treatment of neurotransmitter precursors, etc.: Most PTPS deficiency and DHPR deficiency require a combination of neurotransmitter precursors dopa (levodopa) and 5-hydroxytryptophan. Light PTPS deficiency may not take neurotransmitter precursors [12,14-15].

Levodopa, 5-hydroxytryptophan should start from 1 mg / (kg · d), 1 mg / (kg · d) per week; if possible, according to cerebrospinal fluid neurotransmitter metabolite levels or clinical manifestations Drug treatment dose. Serum prolactin can be used as a reference for dopa dose adjustment, and insufficient dopa dose can also lead to increased prolactin concentration [12,14-15]. In addition, children with DHPR deficiency are prone to secondary cerebral acid deficiency and need to be supplemented with tetrahydrofolate (calcium folinate) 5~20 mg/d [12,14,50].

HPA follow-up and monitoring

Blood Phe concentration: It is recommended to collect Phe concentration after 2 to 3 hours of feeding (infancy) or fasting (after infancy). The blood Phe concentration was measured every 3 days after the start of treatment of special milk powder for children with PKU. The diet was adjusted according to the blood Phe concentration level, and natural food was added. After the metabolic control was stabilized, the Phe measurement time could be adjusted appropriately: <1 year old once a week, 1 ~ 12 years old every 2 weeks ~ once a month, 12 years old and above every 1 ~ 3 months.

If there is an infection, such as an increase in blood Phe concentration, or blood Phe fluctuations, or 3 days after each addition or replacement of the recipe, the blood Phe concentration should be closely monitored. The ideal range of blood Phe concentration control for all ages: 120 ~ 240 μmol / L for 1 year old, 120 ~ 360 μmol / L for 1 ~ 12 years old, and 120 ~ 600 μmol / L for children over 12 years old [8, 10-11].

Prevention of Phe deficiency: Phe is an essential amino acid, excessive or unregulated blood Phe concentration, which may lead to Phe deficiency, severe skin damage, lethargy, anorexia, malnutrition, diarrhea, anemia, hypoproteinemia, etc. Even death [51]. Therefore, it is necessary to strictly monitor the blood Phe concentration, and when the Phe concentration is too low, natural food should be added in time.

Nutrition, physical development, and intelligent development assessment: Height, weight, and nutritional evaluation were measured every 3 to 6 months after treatment to prevent developmental delay and malnutrition. Intelligent development assessment was performed at 1 year, 2 years, 3 years, and 6 years old, and school-age children refer to academic performance [7-8, 10].

Adverse drug reactions: Some patients have gastrointestinal reactions or drug intolerance after taking levodopa and 5-hydroxytryptophan, such as dopa adverse reactions including dyskinesia, involuntary or tic disorder-like movements, excitement and insomnia, etc. Especially in children with initial treatment is easy to occur, reducing dopa dose or total dose can improve the above symptoms; 5-hydroxytryptophan adverse reactions are mainly diarrhea, can be improved after reduction or suspension of drugs; BH4 no obvious Adverse reactions, a small number of headaches, sore throat, diarrhea [12,14].

HPAPrognosis

The prognosis of HPA is related to various factors such as severity of disease, fetal brain development, treatment of morning and evening, blood Phe concentration, nutritional status, and treatment compliance [44, 52-53]. In most patients diagnosed by neonatal screening and starting treatment in the neonatal period, intelligence and physical development can reach or near normal levels, and many patients can normally attend school, employment, marriage, and childbirth.

Reasonable individualized dietary therapy is the key to improving the long-term prognosis of children [28,44-54]. However, even in early screening and early treatment, a small number of patients still lag behind normal children [28, 52-53], and there are problems such as cognitive, mental abnormality or backward social ability in adulthood [55-57].

HPAprevention

Both PAH deficiency and BH4 deficiency are autosomal recessive diseases. The parents of the patients are carriers of disease-causing genes (heterozygotes), and I/4 may be a patient (homozygous) for each birth. The preventive measures have the following aspects [1, 3, 9]:

Avoid close relatives getting married.

Neonatal screening: HPA screening for group blood neonatal Phe detection allows early diagnosis and early treatment of children to avoid or reduce the occurrence of intelligent backwardness [3,7].

Prenatal diagnosis: Informed consent is signed on the premise that the mutations of the proband and their parents are clear, and the disease-related genes are transmitted through placental villi (10-13 weeks pregnant) or amniotic fluid cells (16-22 weeks pregnant). Mutation analysis, to the institution with prenatal diagnostic qualifications for fetal diagnosis and subsequent genetic counseling [1, 58-59].

Author: Chinese Medical moment Sciences Branch of genetic and metabolic endocrinology study group, the Chinese Preventive Medical Association, birth defects prevention and control professional committee newborn screening study group

Article from: Chinese Journal of Pediatrics, June 2014, Volume 52, Issue 6

Writing: Yang Yanling, Ye Jun

Participating in the development of this consensus (in the order of surnames): Ye Jun, Yang Yanling, Zhang Zhixin, Luo Xiaoping, Gu Xuefan, Kong Yuanyuan, Yu Baosheng, Wang Hua, Gong Chunxiu, Zhu Jun, Jiang Jianhui, Sun Qiaoling, Du Minlian, Li Tang, Yang Jianping , Yang Rulai, Qiu Wenjuan, He Yuyu, Zou Hui, Shen Ming, Song Li, Song Fang, Zhang Huiwen, Luo Feihong, Zhao Zhengyan, Ni Guichen, Liang Yan, Liang Li, Han Lianshu, Xiong Hui.