Pyruvate kinase deficiency


Introduction to pyruvate kinase deficiency

Pyruvatekinase (PK) deficiency is a red blood cell enzyme that occurs second only to G-6-PD deficiency.

basic knowledge

The proportion of illness: 0.0005%--0.001%

Susceptible people: no specific people

Mode of infection: non-infectious

Complications: cholelithiasis


Cause of pyruvate kinase deficiency

(1) Causes of the disease

1. Biochemical variant PK is a tetramer with a molecular weight of 60 kD consisting of identical or substantially identical subunits. There are four isomerases in mammalian tissues: L, R, M1 and M2, R-type isomerism. The enzyme (R-PK) is only present in mature red blood cells. R-PK is separated into two components by polyacrylamide gel electrophoresis. Rl-PK is a homotetramer (L2L2), and R1-PK is mainly present in the original. Red blood cells and reticulocytes, while R2-PK is mainly found in mature red blood cells, L-type PK is present in the liver, very similar but not identical to R-PK, M1 type is present in muscle, heart and brain, M2-PK exists in White blood cells and platelets, M2-PK in naive cells, and the presence of M2-PK in red blood cells of some patients with PK deficiency, the heterogeneity of PK mutants can explain the wide range of variability of PK deficiency phenotype, The "classical" PK deficiency, except for the reduction of enzyme activity, has no abnormalities in the characteristics of the other enzymes. At first, it was thought that only the enzymes with normal structure were produced too little, but further studies have shown that there are structural changes in the enzyme molecules that only affect the catalytic activity, obviously Most PK mutations are accompanied by structural abnormal proteins, and These proteins differ in electrophoresis speed, residual activity, substrate affinity, kinetic characteristics, thermal stability, nucleotide specificity, ATP inhibition, allosteric activation or pH optimum.

2. Genetic pattern PK deficiency is autosomal recessive, but occasionally reported as autosomal dominant family. In general, only homozygous or complex heterozygotes will develop hemolytic disease, heterozygous patients despite red blood cells There is a change in glucose intermediates, but no anemia. The detection rate of heterozygous PK deficiency is 0.24% to 2.20%. Most patients with PK deficiency are compound heterozygotes, and there are few true homozygotes.

3. Molecular biology The M2 type PK gene is localized to 15q22-qter, and the L-type and R-type PK genes are located at 1q21. L and R are isoforms regulated by the same gene with two tissue-specific promoters. The encoded L-type and R-type differ only in the first two exons; M1 and M2 are also encoded by the same gene, and two mRNAs respectively translated into this PK are produced due to different splicing. Recently, Kanno et al. The cDNA of human R-type PK gene consists of 2060 bp and encodes a protein consisting of 574 amino acids. PK deficiency is caused by point mutation of PK gene. So far, more than 130 different mutations have been found, mainly missense mutations. A small percentage of patients present with a deletion or insertion.

(two) pathogenesis

The exact hemolysis mechanism of patients with PK deficiency is unclear. When PK is deficient, ATP production is reduced. ATP deficiency is the main cause of hemolysis in PK deficiency. Because of ATP deficiency, it causes the loss of K and water in red blood cells, and the red blood cells shrink. Spinal cells, which have reduced deformability and are retained in the spleen, which are destroyed, leading to the occurrence of sputum anemia, PK deficiency, erythrocyte adenosine diphosphate (ADP) and oxidized coenzyme I (NAD) synthesis impaired, ADP and NAD It will aggravate the decrease of glucose metabolism caused by PK deficiency, thereby aggravating PK, lacking hemolysis in patients, and accumulation of 2,3-diphosphoglycerate (2,3-DPG) in red blood cells of PK deficiency, and 2, 3-DPG is an inhibitor of hexokinase, which also aggravates the decrease in the amount of glucose metabolism caused by PK deficiency, and further decreases the amount of ATP produced to aggravate hemolysis in patients with PK deficiency.


Pyruvate kinase deficiency prevention

Do genetic counseling, check for carriers of disease-causing genes, and give medical guidance on fertility issues.


Pyruvate kinase deficiency complications Complications cholelithiasis

1. Cholelithiasis is a more common complication.

2. Less common complications include bilirubin encephalopathy, chronic leg ulcers, acute pancreatitis secondary to biliary tract disease, spleen abscess, spinal cord compression of extramedullary hematopoietic tissue, and migratory phlebitis.

3. Acute infection or pregnancy can exacerbate the chronic hemolysis process, and even a "hemolytic crisis", which may require blood transfusion.


Symptoms of pyruvate kinase deficiency Common symptoms Red blood cell urinary biliary tract increased hemolytic anemia Astragalus bilirubin increased

Mainly chronic hemolysis and its comorbidities, the severity of the disease may be severe neonatal jaundice, a small number of patients until an adult or old age found anemia, and some due to complete compensation of bone marrow function, usually may not be obvious Anemia and other manifestations, but often have jaundice and spleen in the examination, generally anemia or jaundice occurs in infants or children for the first time, unlike patients with G-6-PD deficiency, PK deficiency infants are always accompanied by anemia when jaundice occurs And often have splenomegaly, the degree of anemia is usually more serious than patients with hereditary spherocytosis, often requiring blood transfusion.

Diagnosis depends on the activity of erythrocyte PK. Care should be taken when considering the diagnosis of PK deficiency:

1 Standardization of fluorescent spot assays for screening PK activity;

2 Except for the possibility of secondary PK deficiency, the following is the diagnostic criteria for PK deficiency.


Examination of pyruvate kinase deficiency

1. Peripheral blood hemoglobin is generally above 50 ~ 60g / L, reticulocyte count is mostly 2.5% ~ 15.0%, after spleen can be as high as 40% ~ 70%, can be seen in peripheral blood can be seen red blood cells and nucleated red blood cells The autologous hemolysis test is non-specific, and this test is no longer used as an experimental diagnostic tool for erythrocyte enzyme disease. Some intermediate products of the glycolysis pathway in red blood cells have characteristic changes, such as 2,3-DPG is twice as large. The above increase, ATP decreased, 3-PG increased, and the like.

2. The PK substrate activity assay method includes fluorescence spot method, PK activity screening test and quantitative determination of PK activity recommended by the International Hematology Standardization Committee. The principle of PK fluorescence point test is to reduce the production of reduced coenzyme I (NADH) in the ultraviolet. Fluorescence can be emitted under light. When tested, phosphoenolpyruvate, NADH and lactate dehydrogenase (LDH) are mixed with the blood to be tested on the filter paper, and the fluorescence intensity is detected. If the blood sample is lacking, NADH is If it is not used, pyruvic acid will not be produced, the fluorescence will last for 45-60 minutes, the normal blood sample will disappear after 15 minutes, and the blood transfusion will lead to false positive. In the application of PK fluorescent spot test, the test should be standardized first, that is, quantified. The results of the calibration method are more reliable. The quantitative determination of PK activity is determined by quantitatively measuring the amount of NADH converted to NAD by spectrophotometer at standard temperature, pH and substrate concentration. When necessary, it is necessary to remove white blood cells as much as possible, because white blood cells contain M1 and M2 type PK enzymes, and the PK activity in white blood cells is 300 times that of normal red blood cells. Leukocytes present in the test sample would lead to false positive, it is generally required leukocyte content of <1.5 × 109 / L.

3. PK substrate activity, inositol-1,6-diphosphate activation and heat stability test. Most of the homozygous or complex heterozygote with anemia showed an enzyme activity level of 5% to 40% of the normal value, and clinical The normal heterozygote has an enzymatic activity of about 50% of normal. For cases of unexplained non-spherical erythrocyte hemolytic anemia, if the PK activity is normal, the activity of PK substrate should be further examined, and the glycoside-1,6-II Phosphoric acid activation and heat stability tests may reveal abnormalities.

According to clinical manifestations, symptoms, signs can choose ECG, B-ultrasound, X-ray and other tests.


Diagnosis and identification of pyruvate kinase deficiency

Diagnostic criteria

1. Normal reference value for PK activity determination

(1) Fluorescence spot method PK activity screening test:

1PK activity was normal: fluorescence disappeared within 25 min.

2PK activity intermediate lack value (hybrid value): fluorescence disappeared at 25 to 60 minutes.

3PK activity was severely deficient (homozygous value): fluorescence did not disappear at 25 min.

(2) Quantitative determination of PK activity [International Hematology Standardization Committee (ICSH)] Recommended Blume method:

1 Normal value: (15.0 ± 1.99) U / gHb (37 ° C).

2 Low substrate concentration (PEP) normal value: 14.9% ± 3.71% (37 ° C) of normal activity.

3 Normal value after low PEP+PDP stimulation: 43.5%±2.46% (37°C) of normal activity.

4 homozygous value is less than 25% of normal activity, and heterozygote value is 25% to 50% of normal activity.

(3) Normal value of intermediate metabolites (37 ° C):

1ATP: (4.23 ± 0.29) mol / g Hb, PK deficiency is more than 2 standard deviations lower than normal.

22,3-diphosphoglycerate (2,3-DPG): (12.27±1.87) mol/g Hb, PK deficiency increased more than 2 times than normal.

3 phosphoenolpyruvate (PEP): (12.2 ± 2.2) mol / LRBC, PK defects increased by more than 2 standard deviations than normal.

42-phosphoglycerate (2-PG): (7.3 ± 2.5) mol / LRBC, PK defects increased by 2 standard deviations than normal.

2. Experimental diagnostic criteria for erythrocyte PK deficiency

(1) The PK fluorescent spot test is a serious lack of value range.

(2) The PK fluorescence spot test is an intermediate lack of value range with a clear family history and/or a 2x increase in 2,3-DPG content or other intermediate product changes.

(3) Quantification of PK activity is a homozygous range.

(4) Quantification of PK activity is a heterozygous range: accompanied by a clear family history and/or intermediate metabolite changes.

In accordance with any of the above 4 items, the experimental diagnosis of PK deficiency can be established. If the clinically highly suspected PK deficiency is present, and the PK activity is normal, the low-substrate PK activity should be quantitatively determined to determine the presence or absence of PK activity. reduce.

3. Diagnostic criteria for hemolytic anemia caused by PK deficiency

(1) Neonatal hyperbilirubinemia caused by erythrocyte PK deficiency:

1 In the early postnatal period (mostly within 1 week), jaundice appeared. The serum total bilirubin of mature children exceeded 205.2 mol/L (12 mg%), and the immature children exceeded 256.5 mol/L (15 mg%), mainly indirect bilirubin. Raise

2 other evidence of hemolysis (such as anemia, increased reticular red, increased urinary biliary, etc.);

3 The diagnostic criteria for PK deficiency meet the above three criteria, and exclude other causes of jaundice, can be diagnosed; those who do not have the above 2 and/or have other reasons, should be suspected of erythrocyte PK deficiency Hemolysis caused by it.

(2) PK deficiency causes congenital non-spherical cellular hemolytic anemia (CNSHA):

1 is a chronic hemolysis process, with splenomegaly, jaundice, anemia;

2 experimental diagnostic criteria in line with PK defects;

3 exclude other erythrocyte enzyme diseases and hemoglobinopathy;

4 Exclusion of secondary PKD, consistent with the above 4 items can be diagnosed as hereditary PKD caused by congenital non-spherical erythrocyte hemolytic anemia.

PK values lower than normal include acute leukemia, MDS, refractory iron granulocyte anemia, and post-chemotherapy status. The cause of acquired enzyme deficiency may be multifactorial. In some cases, it may be accompanied. Bone marrow stem cells with abnormal protein synthesis are damaged, while in other cases, they may be caused by post-translational modifications of the enzyme.

PK deficiency should be differentiated from other erythrocyte enzyme diseases such as G-6-PD deficiency and hemoglobin disease, leukemia, aplastic anemia, myelodysplastic syndrome, and chemotherapy can cause secondary PK deficiency, so hereditary PK deficiency (usually heterozygous) should be differentiated from secondary PK deficiency, but sometimes the identification of the two is quite difficult, because the erythrocyte PK activity is mild to moderately reduced, generally no obvious hemolysis Performance, sometimes need to follow up and carefully analyze.

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