Introduction
Phenylketonuria(phenyl ketonuria, pku) is an inherited disorder in which phenylalanine hydroxylase (pah) deficiency or activity is reduced to cause phenylalanine metabolism. It is more common in hereditary amino acid metabolism-deficient diseases. The genetic pattern of this disease is autosomal recessive inheritance. The clinical manifestations were not uniform. The main clinical features were mental retardation, mental and neurological symptoms, eczema, skin scratch marks, pigmentation and rat odor, and abnormal EEG. If early diagnosis and early treatment are available, the aforementioned clinical manifestations may not occur, intelligence is normal, and EEG abnormalities can be restored.
Cause
(1) Causes of the disease
As the age increases, the amount of phenylalanine ingested for synthetic protein is gradually reduced. After birth, the daily intake of phenylalanine is about 0.5g, and for children and adults it is increased to 4g. The larger part is oxidized to tyrosine, a process that relies mainly on phenylalanine hydroxylase (pah), but also requires cofactor involvement. If this oxidation process is impeded, phenylalanine accumulates in the body. In this case, phenylalanine is metabolized by other means to produce phenylpyruvate harmful substances. Phenylketonuria (pku) is a hereditary disease caused by reduced or absent pah activity. Decreased pah activity also inhibits tyrosine and reduces melanin production, which is inhibited by the accumulation of oxybenzoic acid.
The disease is autosomal recessive, and the mutated gene is located on the long arm of chromosome 12 (12q24.1). The small variation of the gene can cause the disease, not due to gene deletion. It is a hereditary disease caused by the marriage of two heterozygotes. The offspring of close relatives are more common, and about 40% of the children are sick. Due to the mutation of the phenylalanine hydroxylase gene, the phenylalanine hydroxylase deficiency in the liver is a basic biochemical abnormality of the disease. If the base pair of the mutation is different, the severity of the clinical manifestation varies greatly, and it can be expressed as a typical pku or mild hyperphenylalaninemia.
(two) pathogenesis
Phenylalanine (pa) is an essential amino acid that is involved in the formation of various protein components but cannot be synthesized in humans. Under normal circumstances, about 50% of the ingested pa is used to synthesize proteins of various components, and the rest is changed to tyrosine by the action of phenylalanine hydroxylase, and then converted into tyrosine by other enzymes. Dopa, dopamine, adrenaline, norepinephrine and melanin. Phenylalanine hydroxylase is a complex enzyme system. In addition to the hydroxylase itself, it also includes dihydropterin reductase and coenzyme tetrahydrobiopterin. Any enzyme deficiency can cause an increase in blood phenylalanine.
When pa hydroxylase is deficient, phenylalanine that is not involved in the synthesis of the first step protein is accumulated in plasma and deposited in whole body tissues including the brain. The phenylalanine in the blood is discharged beyond the renal threshold, resulting in phenylalanine amino acid urine.
After the main pathway of pa (hydroxylation) is blocked, the secondary metabolic pathway of pa is compensatoryly increased, and the proportion of pa converted to phenylpyruvate, phenyllactate, n-hydroxyphenylacetic acid and phenylacetic acid is gradually increased. Normally, this metabolic bypass is carried out very little, so the content of these metabolites is extremely small; when pa hydroxylase is deficient, these metabolites reach abnormally elevated levels, accumulated in tissues, plasma and cerebrospinal fluid, and a large number. Excreted from the urine, resulting in phenylketonuria.
1. According to the difference of biochemical defects can be divided into
(1) Typical pku: congenital phenylalanine hydroxylase deficiency.
(2) persistent hyperphenylalaninemia: found in phenylalanine hydroxylase isomerase deficiency or heterozygous phenylketonuria, blood phenylalanine increased.
(3) transient mild hyperphenylalaninemia: more common in premature infants, is caused by delayed maturity of phenylalanine hydroxylase.
(4) phenylalanine aminotransferase deficiency: Although the content of blood phenylalanine is increased, phenylpyruvate and hydroxyphenylacetic acid in urine may not be increased, and blood tyrosine is not increased after oral administration of a load of phenylalanine.
(5) Dihydropterin reductase deficiency: complete or partial lack of enzyme activity, in addition to affecting brain development, can make basal ganglia calcification.
(6) Dihydropterin synthesis defects: lack of methanol ammonia dehydratase or other various enzymes.
The typical pku children have normal nervous system at birth. Because of the lack of neuroprotective measures in children with homozygotes, the nervous system is exposed to phenylalanine for a long time. If the mother is homozygous, the blood phenylalanine level is high, the child is heterozygous, the central nervous system damage can occur in the uterus, and the birth manifests as mental retardation.
Ordinary pku and some mild and severe variants, the early stages of the disease can be mentally degraded without treatment. It is speculated that it may be an allelic mutant, manifested as hyperphenylalaninemia, no phenylketonuria and nervous system involvement. In addition, even a small number (about 3%) of patients who control hyperphenylalaninemia cannot prevent progression of neurological disease.
2. Molecular biology studies Normal human pah protein has a fold and has an iron binding site. The maintenance of the iron binding site structure is related to the serine at position 349 in the 3d structure associated with the active site, and the stable polymerization of the serine and pah structures at this site and the catalytic properties of pah are also important. Fusetti et al. determined the crystal structure of human pah (residues 118 to 452) and found that this enzyme and tetramer crystals appeared in each monomeric composition of the catalytic and tetramerization regions. The characteristic in the tetramerization zone is the presence of exchange arms that interact with other monomeric species, thus forming an antiparallel spiral coil, and a significant asymmetry, due to the presence of two chelating zones in the spiral that cause the spiral Caused by an alternating configuration. Some of the most common pah mutations occur at the junction of the catalytic and tetrameric regions.
Mutations in different pah genes cause different degrees of pah activity to be affected, and the effects on pah structure are also different. Camez et al. revealed pah mutations using different expression systems: leu348val, ser349leu, val388met caused pah protein to have folding defects. Expression of the mutated pah protein in Escherichia coli showed thermal instability compared to wild-type pah protein, and the time course of degradation was also different. Bjorgo et al. studied pah7 missense point mutations, namely r252g/q, l255v/s, a259v/t and r270s. There is also a mutation called g272x. When these mutated pah proteins and maltase were co-expressed in Escherichia coli as fusion proteins, the ability to fold and polymerize human pah proteins into homotetramers/dimers was demonstrated to be defective, and most of the recovery was none. Active aggregation type.
R252q and r252g recovered catalytically active tetramers and dimers, and r252g recovered some dimers. The aforementioned three mutations resulted in pah activity of only 20%, 44% and 4.4% of the wild type activity, respectively. When expressed in vitro by a coupled transcription-translation system, all mutant pahs recovered a mixture of non-phosphorylated and phosphorylated forms with low allospecific activity. All of the variant pah proteins expressed by these pah gene mutations are defective in oligomerization, and the sensitivity to restriction protein lysis is increased in vitro, the stability in cells is reduced, and the catalytic activity is also reduced to varying degrees. . All of the foregoing effects appear to be the result of a disordered monomeric structure. Based on the crystal structure of the human pah catalytic domain, the effect of mutation on folding and monomer oligomerization provides an analytical.
These are the correlations between the structural and activity variants of pah protein caused by mutations in the liver pah gene. 99% of hyperphenylalaninemia or pku are caused by mutations in the pah gene, and only 1% are due to disorders in cofactor biosynthesis or regeneration. Mutations in the pah gene may involve exons and introns and may be missense mutations or nonsense mutations.
Mutation types are a bit mutated, inserted or deleted, early stop coding, splicing and polymorphism. The mutated genotypes are homozygous, heterozygous, and complex heterozygotes. Scriver is equivalent to the 1996 review of the pah gene mutation. In 26 countries around the world, 81 researchers analyzed the chromosomes of the 3986 mutation and identified 243 different mutations. By March 1999, zekanowski et al. pointed out in the paper that there are more than 350 pah gene mutations in the world. The authors studied a regulatory region encoding the pah enzyme: a partial exon 3 mutation can cause classical pku, mild pku, and mild hyperphenylalaninemia, the latter mutations often located at positions 71-94. Amino acid residues. Wang Ning pointed out that by April 1998, the global pah gene mutation had increased to 390 species. In China, Xu Lingting et al. reported in 1996 that there are more than 20 mutations in the pah gene, accounting for about 80% of the pah mutant gene. Most scholars believe that there is a correlation between the genotype of the pah mutation and the phenotype, with the exception of a few patients. Guldberg et al. suggest that the inconsistency between the genotype and phenotype of some patients with pah mutations may be due to methods used to examine mutations or due to differences in phenotypic classification.
The pah gene mutations in pku patients in different countries and regions are different, and the distribution of pah gene mutation types in northern and southern populations in China is also inconsistent. The most common mutation in the subgroup of Turkish ancestors was ivs1o-11 g→a (38% of the alleles analyzed); in the Romanian pku patients, the pah gene mutation was mostly arg408trp (47.72% of the allele), lys363fsdelg ( 13.63%) and phe225thr accounted for 6.81%, 3 mutations accounted for 70% of the mutant alleles; arg408trp mutations accounted for 54.9% in Czech pku patients. The differences in the distribution of pah gene mutation types in different regions may reflect multiple mechanisms of pah gene mutation, including founder effect, genetic drift, hypermutability, and selection. .
These are the abnormalities of the pah protein caused by the structure, properties and mutations and mutations of the liver pah gene. In addition to expression in liver cells, pah proteins are also expressed in non-liver tissues, including the kidney, pancreas and brain. The primary structure of pah in the kidney is consistent with that in the liver, except that its regulation is different from pah in the liver, but in the body's phenylalanine balance, the pah of the kidney may work.
In addition to the absence or reduction of liver pah activity can cause pku, there are also changes in the cofactor of pah can also be caused. The main cofactor involved in pah action is 5,6,7,8-tetrahydrobiopterin, which is hydroxylated by phenylalanine, tyrosine and tryptophan. A necessary cofactor. The gene responsible for encoding this substance is the 6-pyruvoyltetrahydropterin synthase (ptps) gene. If the enzyme gene is mutated, ptp is deficient, and pah activity can cause pku even if it is normal. Another enzyme that causes pku is dihydropterin reductase. Accordingly, the pathogenesis of pku involves at least three enzyme genes, and mutation of one gene can cause absent or reduced pah activity, thereby causing pku.
3. Pathological changes in the brain
It manifests as a non-specific change, usually marked by a change in white matter. There are roughly the following situations.
(1) Brain maturity disorders. The fetus begins to have abnormal brain development in the late pregnancy, and the white matter and gray matter stratification of the brain are unclear. There is an ectopic gray matter in the white matter.
(2) Myelin formation disorders. The myelin formation of the cortical spinal cord, cortical-ponsal-cerebellar bundle fibers is most obvious.
(3) gray matter and white matter cystic degeneration; in addition, the dark matter of the brain, the pigmentation of the blue spot disappeared, and the weight of the brain was reduced.
symptom
PKU is a hereditary disease, so neonates have hyperphenylalaninemia. Because they are not eating, the concentration of blood phenylalanine and its harmful metabolites is not high, so there is no clinical manifestation at birth. If the newborn is not screened for phenylketonuria, the phenylalanine and its metabolites in the blood gradually increase with the prolonged feeding time, and the clinical symptoms gradually manifest. The main clinical manifestations are:
1. Growth retardation, in addition to somatic growth and development retardation, is mainly manifested in mental retardation. The performance is lower than the normal baby of the same age, and can appear 4 to 9 months after birth. The intelligence of the heavy-duty is less than 50, and about 14% of the children reach the level of idiots, especially the language development disorder. These manifestations suggest brain developmental disorders. Limiting neonatal intake of phenylalanine prevents intellectual developmental disorders. Mental developmental disorders in children with severe PKU are higher than those in milder blood. It can be considered that mental retardation is related to phenylalanine toxicity, but More detailed pathophysiological mechanisms remain unclear.
2. Neuropsychiatric manifestations There are cerebellar malformations due to brain atrophy, recurrent seizures, but alleviate with age. Increased muscle tone and hyperreflexia. There are often excitement, hyperactivity and abnormal behavior.
3. Skin and hair show that the skin is often dry and prone to eczema and skin scratches. Because of the inhibition of tyrosinase, the melanin synthesis is reduced, so the child's hair is light and brown.
4. Others Due to the lack of phenylalanine hydroxylase, phenylalanine produces increased phenyllactate and phenylacetic acid from another pathway, and is odorous (or murine odor) from sweat and urine.
In general, clinical manifestations and types of PAH gene mutations are associated with the severity of clinical phenotypes, and cofactor deficiency is less clinically phenotypical than PAH protein abnormalities.
diagnosis
Diagnosing the diagnosis of this disease should emphasize early diagnosis in order to get early treatment to avoid mental retardation. Screening for phenylketonuria must be performed in newborns for early diagnosis.
1. Screening Methods The internationally accepted routine screening method is the bacterial inhibition method discovered by Guthrie. Domestic PKU screening kits are available. This method estimates the level of phenylalanine in the blood based on the size of the cultured variability B. subtilis growth zone. If the estimated blood phenylalanine level is 0.24 mmol/L, it is positive. This method can be used for babies 3 to 5 days after birth. Newborns should be screened for newborns with a family history.
2. Phenylalanine load test This test can directly understand the activity of PAH. The loading dose was 0.1 g/kg of oral phenylalanine, and it was taken for 3 days. The blood levels of phenylalanine in children with classic PKU are above 1.22mmol/L, while those with mild type are often below 1.22mmol/L. The latter result suggests that these children may be hyperphenylalaninemia without PKU. .
3. The cause of phenylketonuria caused by the diagnosis of the disease is the PAH gene, and the cause diagnosis is to detect the PAH gene mutation. The detection of PAH gene mutation can not only make an etiological diagnosis for the patient, but also make a prenatal diagnosis for the fetus. There is a correlation between genotype and phenotype in most patients. Different types of mutations have different effects on PAH activity. Therefore, detection of PAH gene mutations is also useful for determining prognosis and guiding treatment.
There are many methods for detecting PAH gene mutations, but one of them is polymerase chain reaction (PCR) combined with one or two of the following detection methods, including single-strand conformation polymorphism (SSCP) and restriction enzyme fragment length. State of the art (RFLP), denaturing gradient gel electrophoresis (DGGE), direct DNA sequencing, mutation-site-specific oligonucleotide probe (ASO), PCR-polyacrylamide gel electrophoresis-silver staining, dideoxy fingerprinting An amplification refractory mutation system (ARMS), an enzyme mismatch lysis method, and the like. The amplified DNA can be analyzed, and the SSCP analysis can also be performed on the RNA. The specimens were analyzed for peripheral blood lymphocytes, and the prenatal diagnosis was performed to analyze polar bodies (gamete products). Analytical polar bodies and ASO can be used for prenatal diagnosis, and the PAH gene of known mutation sites can also be examined by ASO method. There are five most common PAH gene mutations in China: R243Q, Y204C, V399V, Y356X, and R413P. These five PAH gene mutations account for 56.7%. Point mutations are most common in mutations, accounting for 77.4% of the mutation types. Huang Shangzhi proposed a rapid diagnostic procedure for PAH gene mutations: Step 1 for mutation-specific oligonucleotide probe analysis, the diagnostic rate can reach 66%; Step 2 for SSCP analysis of exon 4, the diagnostic rate is increased to 80%; Step 3 Using SSCP analysis to detect several common mutation sites, R243Q (exon 7), V339V and Y356X (exon 11), the diagnostic rate can reach 87%.
The method for detecting the PTPS gene is also based on PCR, combined with the DGGE method, to screen the six coding sequences of this gene and the splice sites of all PTPS genes.
Differential diagnosis of classic and cofactor deficiency caused by PKU patients with hyperphenylalaninemia, but hyperphenylalaninemia does not necessarily cause PKU, so PKU should be compared with other hyperphenylalaninemia Identification.
Transient hyperphenylalaninemia, although the cause of this disease is also due to PAH deficiency, but not due to PAH gene mutation, but PAH immature, resulting in elevated blood phenylalanine concentration of 1.22mmol / L, However, over time, the concentration of blood phenylalanine can be reduced to normal, which can be identified by follow-up blood phenylalanine levels.
Transaminase hyperphenylalaninemia is caused by a lack of phenylalanine aminotransferase. This disease does not cause phenylketonuria. Under normal circumstances, the level of phenylalanine in the blood is normal. Only when eating a high-protein diet, the blood phenylalanine concentration is elevated, and the phenylalanine metabolite level is also normal, so it is associated with PKU. The disease is not difficult to identify.
Light PKU also has only the identification of PKU caused by hyperphenylalaninemia and cofactors. The ratio of phenylalanine to tyrosine can be determined by genetic diagnosis and determination of blood tyrosine level or phenylalanine load test. Identification.
complication
About 2/3 of the children had mild small cranial malformations, normal fundus, no visceral enlargement or abnormal bones.
treatment
PKU is a hereditary disease, and mutations in the PAH gene cannot be cured. However, the serious consequences of hyperphenylalaninemia (ie damage to the brain) can be avoided. The main treatment is to limit the intake of phenylalanine in the diet.
The development of the human brain in the first year after birth (especially the first half of the year) is the most important period, so diet therapy should start from the newborn. The principle of diet therapy is to limit the intake of phenylalanine in the diet and to give enough nutrients for growth and development. If the diet is started 5 months after birth, most of the children have mental retardation; when they start dieting at 4 to 5 years old, they can only reduce the seizures and behavioral abnormalities. As the age increases, the amount of phenylalanine used for protein synthesis will gradually decrease, and the amount of metabolic clearance will gradually increase. If there is a lack of PAH activity, phenylpyruvate is also increasing, so benzene in the diet The amount of alanine should gradually decrease with age.
Generally, the intake of phenylalanine in newborns is 50-70 mg/(kg·d) for 2 months; 40 mg/(kg·d) for 3-6 months; 25-30 mg/(kg·d) for 2 years old; 10~30mg/(kg·d) above the age of. The purpose of diet therapy is to maintain phenylalanine in the blood at 0.24 ~ 0.6mmol / L. The domestically supplied low-phenylalanine foods are benzophenone and benzophenone-based milk powder. Children can be supplemented with breast milk and milk on the basis of this low phenylalanine food feeding.
Each 100 ml of breast milk contains about 40 mg of phenylalanine and 50 mg per 30 ml of milk. Special foods that limit the intake of phenylalanine are expensive and difficult to operate. As for the dietary treatment of limiting phenylalanine intake in the diet, there is no consensus on when to stop. It is generally believed that it must be adhered to for 10 years. Because phenylalanine levels in the blood of patients receiving low phenylalanine diet were lower than those in the rigorous or relaxed diet, phenylalanine levels were lower in blood and IQ was higher (12 months) Treatment results), neuropathy has also been improved, as to when the treatment can relax the diet is not certain.
At the same time as limiting the dietary intake of phenylalanine, the combination of tyrosine supplementation or dietary supplementation with tyrosine to replace phenylalanine in the diet is considered by many scholars to be unnecessary or impossible. Supplementation of tyrosine in the diet can restore hair pigmentation to normal, but has no effect on intellectual progress.
During the dietary treatment that limits phenylalanine intake, the growth, nutritional status, and phenylalanine levels and side effects in the blood should be closely observed. The side effects are mainly other nutritional deficiencies, such as diarrhea, anemia (large cell), hypoglycemia, hypoproteinemia, and niacin-deficient rash.
The above content is for reference only, please consult the relevant physician or relevant medical institution if necessary.
prevention
The treatment of genetic diseases is difficult, the efficacy is not satisfactory, and prevention is more important. Preventive measures include avoiding the marriage of close relatives, conducting genetic counseling, genetic testing of carriers, prenatal diagnosis and selective abortion to prevent the birth of children.
Promote breastfeeding. Early detection of carriers of phenylketonuria and the popularization of ferric chloride diapers make early detection and early treatment of sick infants an important way to prevent mental retardation.
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