Phenylketonuria

Introduction to phenylketonuria Phenylketonuria (PKU) is a hereditary disease caused by phenylalanine hydroxylase (PAH) deficiency or decreased activity of phenylalanine hydroxylase, which is a genetic disorder in hereditary amino acid metabolism. It is more common. The genetic pattern of this disease is autosomal recessive inheritance, and the clinical manifestations are not uniform. The main clinical features are mental retardation, mental and neurological symptoms, eczema, skin scratch marks and pigment loss 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. basic knowledge The proportion of illness: 0.002% Susceptible people: more than children Mode of infection: non-infectious Complications: mental retardation

Etiology of phenylketonuria

Genetic factors (90%):

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 disease, not due to gene deletion, and is caused by the marriage of two heterozygotes. Sexual diseases, the offspring of close relatives are more common, about 40% of children with siblings, due to phenylalanine hydroxylase gene mutation, resulting in a lack of phenylalanine hydroxylase in the liver, this is The basic biochemical abnormalities of the disease, if the base pairs of the mutations are different, cause serious differences in the clinical manifestations, which can be manifested as typical PKU or mild hyperphenylalaninemia.

Pathogenesis

Phenylalanine (PA) is an essential amino acid that is involved in the formation of various protein components, but it cannot be synthesized in the human body. Under normal circumstances, about 50% of the ingested PA is used to synthesize various kinds. The protein of the component, the rest is changed to tyrosine by the action of phenylalanine hydroxylase, and then converted into dopa, dopamine, adrenaline, norepinephrine and melanin by other enzymes, phenylalanine Acid hydroxylase is a complex enzyme system. In addition to the hydroxylase itself, it also includes dihydropterin reductase and the coenzyme tetrahydrobiopterin. Any enzyme deficiency can cause an increase in blood phenylalanine.

When PA hydroxylase is deficient, phenylalanine, which is not involved in the synthesis of the first step protein, is stored in the plasma and deposited in the whole body tissues including the brain, and the phenylalanine in the blood is discharged beyond the renal threshold to produce phenylalanine amino acids. Pee.

After the main pathway of PA (hydroxylation) is blocked, the secondary metabolic pathway of PA is compensatoryly hyperactive, and the specific gravity of PA is converted to phenylpyruvate, phenyllactate, n-hydroxyphenylacetic acid and phenylacetic acid. The metabolic bypass is carried out very little, so the content of these metabolites is very small; when the PA hydroxylase is deficient, these metabolites reach an abnormally high level, accumulated in tissues, plasma and cerebrospinal fluid, and a large amount from the urine. Excreted to produce 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. Children are heterozygous, and central nervous system damage can occur in the uterus, which is manifested as mental retardation at birth.

Ordinary PKU and some mild and severe variants, the early stage of the disease can be mentally degraded without treatment, presumably an allelic mutation, manifested as hyperphenylalaninemia, no phenylketonuria And the nervous system is involved, in addition, even a small number (about 3%) of patients control hyperphenylalaninemia, can not prevent the progression of neurological diseases.

2. Molecular biology studies The normal human PAH protein has a fold and has an iron binding site. The retention of the iron binding site structure is related to the serine at position 349 located in the 3D structure associated with the active site. The stable polymerization of serine and PAH structures 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 each of these enzymes and constituent catalytic and tetramerization zones Monomers appear as tetramer crystals, and the tetramerization zone is characterized by the presence of exchange arms that interact with other monomeric species, thus forming an antiparallel spiral coil, and is significantly asymmetrical due to The chelating region that causes the spiral of the spiral is caused by two alternating configurations, some of which 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 with different expression systems: Leu348Val, Ser349Leu, Val388Met cause folding defects in PAH protein, which will be mutated. The expression of PAH protein in Escherichia coli showed heat instability compared with wild-type PAH protein, and the time course of degradation was different. Bjorgo et al. studied PAH 7 kinds of missense point mutations, namely R252G/Q, L255V/S, A259V. /T and R270S, another mutation is G272X. When these mutant PAH proteins are co-expressed with maltase as a fusion protein in Escherichia coli, it is proved that human PAH protein is folded and polymerized into the same tetramer/ The ability of dimers is defective, most of them are inactive aggregates, R252Q and R252G recover catalytically active tetramers and dimers, R252G recovers some dimers, and the above three mutations cause PAH activity Only 20%, 44%, and 4.4% of wild-type activity, when expressed in vitro by a coupled transcription-translation system, all mutant PAHs recovered non-phosphorus with low allospecific activity A mixture of chemokines and phosphorylated forms, all of the variant PAH proteins expressed by mutations in the PAH gene are defective in oligomerization, increased sensitivity to restriction protein lysis in vitro, and reduced stability in cells. The catalytic activity is also reduced to varying degrees. All of the foregoing effects appear to be due to the disorder of the monomer structure. According to the crystal structure of the human PAH catalytic region, the effect of the mutation on the folding and monomer oligomerization provides an analysis. .

These are the correlations between PAH protein structure and activity variation caused by some PAH gene mutations. 99% of hyperphenylalaninemia or PKU are caused by PAH gene mutation, only 1% is due to cofactor biosynthesis or Regeneration is caused by disorders. PAH gene mutations may involve exons and introns. They may be missense mutations or nonsense mutations. The mutation types are a little mutated, inserted or deleted, coding stops early, splicing and polymorphism, and mutations. Genotypes are homozygous, heterozygous and complex heterozygote. Scriver is equivalent to the PAH gene mutation reviewed 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 PAH gene mutations in the world. The author studied the PAH enzyme regulatory region: part of the exon 3 mutation can cause classic PKU, mild PKU. And mild hyperphenylalaninemia, the latter mutation is often located in the amino acid residues 71-94, Wang Ning pointed out that by April 1998, the global PAH gene mutation has increased to 390, in our country 1996 Xu Ling Other reports have identified more than 20 PAH mutations, 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. Inconsistencies between genotypes and phenotypes of PAH mutations 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 is IVS1O-11 GA (according to the analyzed alleles). 38% of genes); PAH gene mutations in Romanian PKU patients were mostly Arg408Trp (47.72% of alleles), Lys363fsdelG (13.63%) and Phe225Thr accounted for 6.81%, and 3 mutations accounted for 70% of mutant alleles; The Arg408Trp mutation in PKU patients accounted for 54.9%. The distribution of PAH gene mutation types in different regions may reflect multiple mechanisms of PAH gene mutation, including founder effect, genetic drift, and excessive swapping. Hypermutability and selection.

The above are the PAH gene structure, nature and mutations and mutations caused by PAH protein abnormalities. PAH protein is expressed in non-liver tissues, including kidney, pancreas and brain, and PAH level in the kidney. The structure 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 lack or decrease of liver PAH activity can cause PKU, there are also changes in cofactors of PAH, and the main cofactor involved in PAH action is 5,6,7,8-tetrahydrobiopterin (5,6). ,7,8-tetrahydrobiopterin), a substance necessary for the hydroxylation of phenylalanine, tyrosine and tryptophan. The gene responsible for encoding this substance is 6-pyruvyltetrahydropterin synthase (6- Pyruvoyltetrahydropterin) synthase (PTPS) gene, if the enzyme gene is mutated, PTP is deficient, PAH activity can cause PKU even if it is normal, and another enzyme that causes PKU is dihydropterin reductase. 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 is characterized by non-specific changes, usually white matter changes are obvious, and there are roughly the following cases.

(1) Brain maturity disorder, the fetus begins to have abnormal brain development in the late pregnancy, the white matter of the brain, the stratification of the gray matter is unclear, and there is an ectopic gray matter in the white matter.

(2) Myelin formation disorders, the most obvious is the myelin formation of the optic tract, corticospinal tract, cortical-ponsal-cerebellar bundle fibers.

(3) gray matter and white matter cystic degeneration; in addition, there are substantia nigra of the brain, the pigmentation of the blue spot disappears, and the weight of the brain is reduced.

Phenylketonuria prevention

(1) Gradually and comprehensively promote the screening of phenylketonuria in the neonatal period. Early detection of children with phenylketonuria was detected in heterozygous families, avoiding close relatives' marriage, heterozygous should not be married, and genetic counseling should be done. To guide family planning and reduce the birth rate of patients with phenylketonuria. For families with existing children, prenatal diagnosis should be carried out when they are born again, that is, fetal villi or amniotic fluid is taken in the early or middle part of pregnancy, and genetic diagnosis is performed by recombinant DNA technology to diagnose whether the fetus is a normal child, a carrier or a child. This makes the decision to continue or terminate the pregnancy.

(2) pregnant women should limit the intake of phenylalanine, if the blood phenylalanine concentration exceeds 726.4-908 / mol / L should be treated, so that the blood concentration is maintained at 363.2 - 484.3mol / L, the concentration is too low Or phenylalanine deficiency can also cause fetal damage. Provide enough protein during pregnancy, the minimum daily amount is 75-80g.

(3) Promote breastfeeding, find carriers and children with phenylketonuria as early as possible, and start treatment early to prevent mental retardation. Popularize measures such as ferric chloride diapers.

Phenylketonuria complications Complications, mental retardation

About 2/3 of the children had mild small cranial malformations, normal fundus, no visceral enlargement or abnormal bones.

Symptoms of phenylketonuria common symptoms phenylalanine metabolic disorder mental retardation hair pale and brown repeatedly twitching cerebellar malformation eczema

PKU is a hereditary disease, so neonates have hyperphenylalaninemia. Because they are not fed, the concentration of blood phenylalanine and its harmful metabolites is not high, so there is no clinical manifestation at birth. Children were not screened for phenylketonuria. As the feeding time prolonged, the phenylalanine and its metabolites in the blood gradually increased, and the clinical symptoms gradually showed up. The main clinical manifestations were:

1. Growth retardation: In addition to somatic growth and development retardation, it is mainly manifested in mental retardation, which is manifested in lower IQ than normal infants of the same age. It can occur 4 to 9 months after birth, and the IQ of heavy ones is less than 50, about 14%. The above children reach the level of idiots, especially the language development disorder. These manifestations suggest brain developmental disorders, restricting neonatal intake of phenylalanine to prevent mental retardation, and mental developmental disorders in children with severe PKU compared with lighter phenylalanine. The high concentration, according to which can be considered that mental retardation is related to phenylalanine toxicity, but the detailed pathophysiological mechanism remains unclear.

2. Neuropsychiatric manifestations: There are cerebellar malformations due to brain atrophy, recurrent convulsions, but with the increase of age, muscle tension is increased, hyperreflexia, often have excitement, hyperactivity and abnormal behavior.

3. Skin and hair performance: The skin is often dry, prone to eczema and skin scratches. Because of the inhibition of tyrosinase, the synthesis of melanin is reduced, so the hair of the child is pale and brown.

4. Others: Due to the lack of phenylalanine hydroxylase, phenylalanine produces phenyllactate and phenylacetic acid from another pathway, which is excreted from sweat and urine and has a mildew odor (or rat smell).

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.

Examination of phenylketonuria

1. Uric phenylpyruvate test: Due to the increase of phenylpyruvate in the urine of children, qualitative tests can be carried out. The methods are as follows:

(1) Ferric chloride test: 5% ferric chloride was instilled into 5 ml of urine, and the green reaction immediately became positive. The newborn was not fed, and the test was negative. The urine of the diabetic can also be positive, so the test Poor specificity.

(2) 2,4-nitrophenylhydrazine test: positive if a yellow turbid precipitate is produced.

2. Determination of blood phenylalanine: normal human blood phenylalanine is 60 ~ 180mol / L, PKU patients can be as high as 600 ~ 3600mol / L, if 258mol / L is the dividing point between normal and PKU patients, then There are up to 4% false positives. Color chromatography can cause false negatives in newborns after a few days of life. MS/MS can reduce false positive rate. This method can simultaneously measure blood phenylalanine and tyrosine. Acid, and can calculate the ratio of phenylalanine / tyrosine. If the ratio of 2.5 is the cut-off point between normal children and those with PKU, the false positive can be reduced to 1%. Therefore, this method is currently used to screen neonatal benzene. Acetoneuria, this method can also be used to screen for galactosemia, maple diabetes, homocystinuria and congenital hypothyroidism. A single examination can screen a variety of congenital diseases.

3. Electroencephalogram (EEG): mainly spine slow wave, occasionally high amplitude rhythm disorder, EEG follow-up study showed that with the increase of age, EEG abnormal performance gradually increased, and EEG abnormalities gradually decreased after 12 years old.

4. Prenatal examination: Since the villous and amniotic fluid cells can not detect the activity of phenylalanine hydroxylase, the prenatal diagnosis problem can not be solved for a long time. At present, 25 Chinese PKU disease-causing gene mutations have been identified in China, accounting for about 80% of the phenylalanine hydroxylase mutant gene in China has been successfully used in the detection of mutated mutations and prenatal diagnosis in patients with PKU.

5. X-ray examination: visible microcephaly, CT and MRI can find non-specific changes such as diffuse cortical atrophy.

Diagnostic identification of phenylketonuria

Diagnostic criteria

The diagnosis of this disease should emphasize early diagnosis in order to get early treatment to avoid mental retardation, and screening for phenylketonuria must be performed in neonates for early diagnosis.

1. Screening method: The internationally recognized routine screening method is the bacterial inhibition method discovered by Guthrie. The domestic PKU screening kit is available. This method is based on the growth of the variability of B. subtilis growth band. To estimate the level of phenylalanine in the blood, if the estimated blood phenylalanine level is positive at 0.24mmol / L, this method can be used for babies 3 to 5 days after birth, for newborns with a family history More neonatal screening should be performed.

2. Phenylalanine load test: This test can directly understand the activity of PAH, the load dose is oral phenylalanine 0.1g / kg, and even served for 3 days, the classic PKU children with blood phenylalanine level at 1.22 Above mmol/L, mild cases 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 PAH gene. The etiological diagnosis is to detect PAH gene mutation. The detection of PAH gene mutation can not only diagnose the cause of the patient, but also make prenatal diagnosis for the fetus, genotype. There is a correlation between phenotype 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 the length of restriction enzyme fragments is large. 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), enzyme mismatch cleavage method, etc., can analyze the amplified DNA, can also perform SSCP analysis on RNA, analyze peripheral blood lymphocytes for specimens, prenatal diagnosis The polar body (gamete product) can be analyzed, and the polar body and ASO can be used for prenatal diagnosis. The PAH gene of known mutation site can also be examined by ASO method. There are five kinds of PAH gene mutations in China: R243Q, Y204C, V399V, Y356X, R413P, these 5 PAH gene mutations accounted for 56.7%, the most common point mutations in the mutation, accounting for 77.4% of the mutation type, Huang Shangzhi proposed a rapid diagnosis procedure for PAH gene mutation: Step 1 for the mutation point Specific oligonucleotide probe analysis, diagnostic rate Up to 66%; Step 2 for SSCP analysis of exon 4, the diagnostic rate increased to 80%; Step 3 uses SSCP analysis to detect several common mutation sites, namely 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

Patients with PKU caused by classic and cofactor deficiency have hyperphenylalaninemia, but those with hyperphenylalaninemia do not necessarily cause PKU, so PKU should be differentiated from other hyperphenylalaninemia patients. .

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, in general, the level of phenylalanine in the blood is normal, only when eating high protein diet blood benzene The concentration of alanine is elevated and the level of phenylalanine metabolites is normal, so it is not difficult to identify with PKU.

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.

The material in this site is intended to be of general informational use and is not intended to constitute medical advice, probable diagnosis, or recommended treatments.