Phenylalanine metabolism disorder

Introduction Phenylketonuria (PKU) is a hereditary disease caused by a deficiency or loss of activity of phenylalanine hydroxylase (PAH) in the liver. 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 primarily 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 an inherited disease caused by reduced or absent PAH activity. Decreased PAH activity also inhibits tyrosine and reduces melanin production, and hydroxyphenylpyruvase is inhibited to cause hydroxybenzoic acid to accumulate in the body.

The disease is autosomal recessive, and the mutated gene is located on the long arm of chromosome 12 (12q24.1). The small mutation of this 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 manifestations varies greatly, and may be manifested 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 various components of the protein, and the rest is converted 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 specific gravity of PA is converted into phenylpyruvate, phenyllactate, n-hydroxyphenylacetic acid and phenylacetic acid. 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 research

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 118-452) and found that this enzyme and tetramer crystallization appeared in each monomeric composition of the catalytic and tetramerization zones. 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 have different effects on PAH activity and have different effects on PAH structure. Camez et al. revealed PAH mutations using different expression systems: Leu348Val, Ser349Leu, Val388Met caused folding defects in PAH proteins. Expression of the mutated PAH protein in Escherichia coli showed thermal instability compared to the wild-type PAH protein, and the time course of degradation was also different. Bjorgo et al. studied PAH 7 missense point mutations, namely R252G/Q, L255V/S, A259V/T and R270S. There is also a mutation called G272X. When these mutated PAH proteins were co-expressed with maltase as a fusion protein in Escherichia coli, 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 recover catalytically active tetramers and dimers, and R252G recovers 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 mutated 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 region, the effect of mutation on folding and monomer oligomerization provides an analytical.

These are the correlations between PAH protein structure and activity variation 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 et al. reviewed the PAH gene mutation in 1996. In 26 countries around the world, 81 researchers analyzed 3986 mutant chromosomes and identified 243 different mutations. By March 1999, Zekanowski et al. pointed out in the paper that there are more than 350 mutations in the PAH gene in the world. The authors studied a PAH-encoding regulatory region: a partial exon 3 mutation can cause classical PKU, mild PKU, and mild hyperphenylalaninemia, with mutations often located between 71 and 94. Amino acid residues. Wang Ning pointed out that by April 1998, the number of PAH gene mutations in the world had increased to 390. In China, Xu Lingting and other reports in 1996 have identified 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 PAH mutations in some patients may be due to methods used to examine mutations or due to differences in phenotypic classification.

The PAH gene mutations of PKU patients in different countries and regions are different. The distribution of PAH gene mutation types in northern and southern China is also inconsistent. The most common mutation in the subgroup of Turkish ancestors was IVS1O-11 GA (38% of the alleles analyzed); in the PKU patients in Romania, the PAH gene mutation was mostly Arg408Trp (accounting for 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 play a role.

In addition to the absence or reduction of liver PAH activity can cause PKU, and changes in cofactors of PAH can also be caused. The main cofactor involved in the action of PAH 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 even if the PAH activity is normal, PKU can be caused. Another enzyme that causes PKU is dihydropterin reductase. Accordingly, the pathogenesis of PKU involves at least three enzyme genes, one of which can cause a deficiency or decrease in PAH activity, resulting in 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.

an examination

Related inspection

Serum phenylalanine phenylalanine deaminase test

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 method

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. Etiology diagnosis

The gene causing phenylketonuria is the PAH gene, and the etiological 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 and combined with the DGGE method to screen the six coding sequences of the gene and the splice sites of all PTPS genes.

Differential diagnosis

Complex urinary tract infection: Complex urinary tract infection refers to: 1 urinary tract has organic or functional abnormalities, causing urinary tract obstruction, poor urinary flow; 2 urinary tract has foreign bodies, such as stones, indwelling catheters, etc. 3 renal obstruction, such as urinary tract infections on the basis of chronic renal parenchymal disease, mostly pyelonephritis, can cause kidney damage. Long-term repeated infection or incomplete treatment can progress to chronic renal failure (CRF).

Specific amino acid urinary: mainly threonine serine histidine alanine hydroxyproline excretion is normal, so it can be distinguished from all amino acid urinary glycine valine and hydroxyproline excretion can be distinguished from iminoglycine urine The excretion of two base amino acids in the urine is also normal to be distinguished from cystine urine.

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.

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