What Is Pyruvate Kinase Deficiency?

In 1953, Dacie et al. First described a group of heterogeneous diseases called congenital non-spherical cell hemolytic anemia. In 1954, Selwgn and Daice divided this group of diseases into two types according to the autohemolysis test. Type I autohemolysis can only be corrected after a slight increase in glucose, and type II autohemolysis can be significantly increased. Adding glucose can not correct De Gruchy and others in 1960. It was found that patients with type can be corrected after adding adenosine triphosphate (ATP), which indicates that their ATP production is impaired and its essence is pyruvate kinase (PK) deficiency. In 1961, Valentine first confirmed PK deficiency in red blood cells in patients with type II congenital non-spherical hemolytic anemia, the frequency of which was second only to G-6-PD deficiency.

Pyruvate kinase deficiency

Chinese name: pyruvate kinase deficiency English name: pyruvate kinase deficiency Pyruvate kinase (PK) deficiency is a red blood cell enzyme disease that occurs second only to G-6-PD deficiency. It has been confirmed that PK deficiency is caused by PK gene abnormality, mainly because a small number of patients with PK gene point mutations show deletion or insertion of ATP deficiency, which is the main factor for the diagnosis of this disease. There is no specific treatment for this disease

- Pyruvate Kinase Deficiency -Overview

- Pyruvate kinase deficiency Pyruvate kinase deficiency-epidemiology

Autosomal recessive inheritance is high in people of Nordic descent. The disease is roughly equal to the number of G-6PD deficiency in Japan, but there is growing evidence that the disease is also globally distributed. Hemolysis due to PK deficiency has been seen in Portugal, Italy, Near East, Australia and New Zealand, Venezuela, China, Philippines, Mexico and other regions and countries. Hong Kong, China, 3% of newborns are PK variant heterozygotes. Zygote is about 1%. China's Hu Yamei reported two cases for the first time in 1984 in China. So far (before May 2012), 12 cases of hemolytic anemia caused by PK deficiency were reported in China.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-etiology

1. Biochemical variant PK is a tetramer with a molecular weight of 60kD consisting of identical or substantially identical subunits. There are four isomerases in mammalian tissue: LR M1 and M2 R-isomerase (R-PK R-PK, which is only found in mature red blood cells, is divided into two components after polyacrylamide gel electrophoresis. Rl-PK is a homotetramer (L2L2). R1-PK is mainly found in primitive red blood cells and reticulocytes, and R2-PK is mainly found in mature red blood cells. L-type PK is present in the liver, and is very similar but not identical to R-PK. M1 type is found in muscle heart and brain, M2-PK is found in white blood cells and platelets, and M2-PK is also found in naive cells. In some patients with PK deficiency, the presence of M2-PK has been found in the red blood cells. The heterogeneity of the PK mutant can explain the wide range of PK deficiency phenotypes. The "classical" PK lacks enzymes other than reduced enzyme activity. At first, it was thought that only the normal structure of the enzyme was too little, but further research proved that there were changes in the molecular structure of the enzyme that only affected the catalytic activity. Obviously, most of the PK mutations are accompanied by structural abnormal proteins, and these proteins are different in electrophoretic speed residual active substrate affinity kinetics characteristics, thermostability nucleotide-specific ATP inhibition allosteric activation, or optimal pH.

2. Genetic pattern PK deficiency is reported as an autosomal recessive but occasionally an autosomal dominant family. Generally, only homozygous or complex heterozygotes will cause hemolytic disorders. Heterozygous patients showed no anemia despite changes in glucose intermediates in red blood cells. The detection rate of heterozygosity for PK deficiency is 0.24% to 2.20%. Most patients with PK deficiency have few heterozygotes.

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

- Pyruvate Kinase Deficiency Pyruvate Kinase Deficiency-Pathogenesis

The exact mechanism of hemolysis in patients with PK deficiency is unclear. In the absence of PK, ATP production is reduced. ATP deficiency is the main cause of hemolysis due to PK deficiency. When ATP is lacking, Na ions accumulate in red blood cells, red blood cells swell into a spherical shape, and spherical red blood cells are destroyed as they pass through the spleen, leading to the occurrence of hemorrhagic anemia. PK deficiency Erythrocyte adenosine diphosphate (ADP) and oxidized coenzyme I (NAD +) synthesis impaired ADP and NAD + can aggravate the decrease in glucose metabolism due to PK deficiency and thereby increase hemolysis in patients with PK deficiency In addition, PK deficiency red blood cells 2 3-diphosphoglycerate (2,3-DPG) accumulates, and 2 3-DPG is an inhibitor of hexokinase. This also exacerbates the decrease in glucose metabolism caused by PK deficiency. The decrease in ATP production further reduces PK deficiency. The patient's hemolysis worsened.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-clinical manifestations

Mainly the manifestations of chronic hemolysis and its comorbidities. The severity of the disease can be severe neonatal jaundice or even bilirubin encephalopathy, which requires blood replacement or multiple blood transfusions. A few patients do not find anemia until adulthood or old age, and they are completely compensated for bone marrow function. Obvious anemia and other manifestations but jaundice and splenomegaly are often found on examination. Generally, anemia or jaundice first occurs in infants or children. Unlike patients with G-6-PD deficiency, infants with PK deficiency always have anemia and often have splenomegaly when they develop jaundice. The degree of anemia is usually more than that of hereditary spherical red blood cells More severe cases often require blood transfusions.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-complications

1. Cholelithiasis is a more common complication.

2. Rare complications include chronic leg ulcers with bilirubin encephalopathy, acute pancreatitis secondary to biliary disease, spleen abscess, spinal cord compression of extramedullary hematopoietic tissue, and walking phlebitis, etc.

3. Acute infection or pregnancy can exacerbate the chronic hemolytic process, and even a "hemolytic crisis" may occur at this time.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-diagnosis

Diagnosis depends on the determination of PK activity of red blood cells. When considering the diagnosis of PK deficiency, attention should be paid to: standardization of the fluorescent spot test for screening for PK activity; the possibility of secondary PK deficiency is excluded, the following are the diagnostic criteria for PK deficiency.

1 Normal reference value for PK activity determination

(1) Screening test for PK activity by fluorescent dot method:

PK activity is normal: fluorescence disappears within 25min

Intermediate deficiency of PK activity (hybrid value): fluorescence disappears at 25 to 60 minutes

Severe lack of PK activity (homozygosity value): fluorescence does not disappear at 25min

(2) Quantitative determination of PK activity [Blume method recommended by the International Committee for Standardization of Hematology (ICSH)]:

Normal value: (15.0 ± 1.99) U / gHb (37 )

Normal value of low substrate concentration (PEP): 14.9% ± 3.71% of normal activity (37 )

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

The homozygous value is less than 25% of the normal activity, and the heterozygous value is 25% to 50% of the normal activity

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

ATP: (4.23 ± 0.29) mol / gHb, which is more than 2 standard deviations lower than normal in the absence of PK

Glycerol 2,3-diphosphate (2 3-DPG): (12.27 ± 1.87) mol / gHb PK deficiency increased more than twice as much as normal.

Phosphoenolpyruvate (PEP): (12.2 ± 2.2) mol / LRBC, when the PK defect is increased by more than 2 standard deviations.

2-phosphoglycerate (2-PG): (7.3 ± 2.5) mol / LRBC PK deficiency increased by 2 standard deviations from normal.

2. Laboratory diagnostic criteria for erythrocyte PK deficiency

(1) The PK fluorescence spot test is a severely lacking value range.

(2) The PK fluorescence spot test is in the middle of the lack of value range, with a clear family history and (or) 2-3-DPG content increased more than 2-fold or other intermediate products change.

(3) Quantitative PK activity belongs to the homozygous range.

(4) Quantitative PK activity is in the heterozygous range: with a clear family history and / or changes in intermediate metabolites

If any one of the above 4 items is met, an experimental diagnosis of PK deficiency can be established. If clinically suspected of PK deficiency, and when the PK activity is normal, a quantitative measurement of low substrate PK activity should be performed to determine whether there is a decrease in PK activity.

3 diagnostic criteria for hemolytic anemia caused by PK deficiency

(1) Hyperbilirubinemia in neonates due to erythrocyte PK deficiency: The serum total bilirubin of mature children with jaundice in the early postnatal period (mostly within 1 week) exceeds 205.2 mol / L (12mg%) of immature children Exceeding 256.5mol / L (15mg%) is mainly indirect bilirubin elevation; other evidence of hemolysis (such as anemia, reticulocyte increase, urobilinogen increase, etc.); meet the experimental diagnostic criteria for PK deficiency. Those who have the above three items and exclude other causes of jaundice can be diagnosed; those who do not have the above two items and / or coexist with other reasons should be suspected to be diagnosed as hemolysis caused by PK deficiency of red blood cells.

(2) Congenital non-spherical cell hemolytic anemia (CNSHA) due to PK deficiency: chronic hemolytic process with spleen jaundice and anemia; meet the experimental diagnostic criteria for PK deficiency; exclude other red cell enzyme diseases and hemoglobin Disease; exclude secondary PKD in accordance with the above 4 before diagnosis of congenital non-spherical erythrocyte hemolytic anemia caused by hereditary PKD.

Diseases with a lower than normal PK value also include acute leukemia, refractory iron granulocyte anemia, and state-acquired enzyme deficiency after chemotherapy may be multifactorial, and in some cases may be accompanied by protein Abnormally synthesized bone marrow stem cells are damaged and, in other cases, may be caused by post-translational modifications of the enzyme.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-differential diagnosis

PK deficiency should be distinguished from other red cell enzyme diseases such as G-6-PD deficiency and hemoglobin disease. Leukemia, aplastic anemia, myelodysplastic syndrome can cause secondary PK deficiency after chemotherapy, so hereditary PK deficiency (usually heterozygous) should be distinguished from secondary PK deficiency, but sometimes both The identification is very difficult because the erythrocyte PK activity of both is slightly to moderately reduced, and generally there is no obvious hemolytic manifestation, and sometimes follow-up visits and careful analysis are required.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-laboratory test

1. Peripheral hemoglobin is generally 50 60g / L, and the net red blood cell count is mostly 2.5% 15.0%. After cutting the spleen, it can reach as high as 40% 70%. Spinal red blood cells and nucleated red cells can be seen in peripheral blood. The autohemolytic test is non-specific, and this test is no longer used as an experimental diagnostic method for erythrocyte enzyme disease. Some intermediate products of glycolysis pathway in red blood cells have characteristic changes, such as 2-3-DPG shows more than 2-fold increase, ATP decreases, 3-PG increases, etc.

2 PK substrate activity measurement methods include fluorescence dot method, PK activity screening test and Blume method for quantitative determination of PK activity recommended by the International Committee for Standardization of Hematology. The principle of PK fluorescence point test is reduction to produce reduced coenzyme (NADH) under ultraviolet light. It can emit fluorescence for the test. When phosphoenolpyruvate NADH and lactate dehydrogenase (LDH) are mixed with the test blood added to the filter paper and incubated, the fluorescence intensity is measured. If the blood sample lacks PK, NADH will not be produced without pyruvate, and the fluorescence will continue for 45-60 minutes. Normal blood samples, 15 minutes after the disappearance of fluorescence can lead to false positives after blood transfusion. In the application of the PK fluorescence spot test, the test should be standardized first, that is, the result of the screening method is corrected by a quantitative method, so that the result is more reliable. Quantitative measurement of PK activity is determined by quantitatively measuring the amount of NADH converted to NAD with a spectrophotometer at standard temperature, pH and substrate concentration. When determining the PK activity of red blood cells, it is necessary to remove white blood cells as much as possible because white blood cells contain M1. The PK activity in white blood cells and M2 type PK enzymes is 300 times that of normal red blood cells. The presence of white blood cells in the test sample will lead to false positives, so the white blood cell content is generally required to be less than 1.5 × 109 / L.

3 PK substrate activity, methylose-1,6-bisphosphate activation and thermostability tests Most homozygous or complex heterozygotes with anemia manifest their enzyme activity levels at 5% to 40% of normal values, while clinically normal The heterozygous enzyme activity is about 50% of normal. For cases of non-spherical erythrocyte hemolytic anemia of unknown origin, if the PK activity is normal, the PK substrate activity, methylose-1 6-diphosphate activation, and Thermal stability tests may find abnormalities.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-other auxiliary tests

According to clinical manifestations, symptoms, and signs, the ECG B-X-ray can be selected.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-related tests

· Glyceryl 2,3 diphosphate

·> PH (urine)

·> Pyruvate

·> Pyruvate kinase

·> Lactate dehydrogenase

·> Prostaglandins

·> Antidiuretic hormone

·> Nucleated red blood cells

·> Spiny red blood cells

·> Spherical red blood cells

·> Erythrocyte 3-phosphoglycerate

·> RBC -phosphoglycerate

·> Erythrocyte adenosine triphosphate

·> Average red blood cell volume

·> Erythrocyte phosphoenolpyruvate

·> Red blood cell count

·> Autolysis Test

·> Hemoglobin

- Pyruvate kinase deficiency Pyruvate kinase deficiency-treatment

1. Blood transfusion In the first few years after birth, the best treatment for severe anemia is to maintain the hemoglobin concentration of red blood cell transfusion above 80-100g / L without affecting the growth and development of children, and reduce the risk of life-threatening aplastic anemia. It is important to base the patient's tolerance on anemia and not just the level of hemoglobin. Because the patient's red blood cell 2 3-DPG level is increased, there is no obvious discomfort in moderate to severe anemia.

2. Splenectomy splenectomy can make the patient control anemia for a long time. Because there is a risk of severe sepsis in the spleen-free state in the first few years after birth, the patient should be at least 5 to 10 years old after splenectomy. Splenectomy can improve the prognosis, but it does not correct the hemolytic status. Those who need blood transfusion before surgery may not need to be infused after surgery. Younger children have improved exercise tolerance during the catch-up period of rapid hematopoietic growth. Although the possibility of aplastic anemia cannot be completely ruled out, it is usually milder after the occurrence, and after the initial improvement, Hb may gradually decrease. An increase in the number of reticulocytes in a patient after surgery indicates that an incomplete compensatory hemolytic process persists. In the selection of patients for splenectomy, the preoperative evaluation of red blood cell survival and spleen blood volume is not significant because some patients' liver is the main site of red blood cell destruction. The spleen seems to destroy more severe red blood cells. In short, the more severe the anemia, the more severe the spleen. The better the removal effect

3. The clinical significance of the phenomenon that salicylate adversely affects the energy metabolism of PK-deficient cells in vitro once drug treatment can be applied under strict hematological monitoring. Severe PK deficiency has also been observed. Female patients have increased hemolysis when taking oral contraceptives

4. Patients with severe hemolytic anemia caused by PK deficiency due to allogeneic bone marrow transplantation (Allo-BMT) or peripheral blood stem cell transplantation (Allo-PBSCT) or cord blood transplantation. If repeated blood transfusions are required to sustain life, Allo-BMT or Allo- PBSCT is the only cure.

- Pyruvate kinase deficiency Pyruvate kinase deficiency-prognosis

The prognosis is inconsistent due to the severity of the disease. Infants and young children can cause death. The disease tends to weaken with age. Most patients can lead a relatively normal life with no significant effect on life expectancy.