RU2187812C1 - Method for predicting the development of iron deficiency anemia (variants) - Google Patents

Abstract

FIELD: medicine, hematology. SUBSTANCE: it is conducted the analysis of polymorphous loci of P-4501A1 cytochromic gene due to polymerase chain reaction of DNA synthesis, it is conducted in DNA isolated due to phenol-chloroform extraction technique out of lymphocytes of peripheral venous blood. After amplification and extraction DNA fragments are electrophoretically divided in 7%- polyacrylamide nondenaturated gel and analyzed under ultraviolet lighting by using a transulluminator: the presence of DNA-fragments at the size of 48 and 139 nucleotide pairs (np) is interpreted as isoleucin allele (Ile), at the size of 48, 120 and 19 np - as valine (Val). Relative risk of disease development (RR) is concluded on at RR>1.5. At revealing Ile/Val genotype it is predicted the risk of IDA development (variant 1). Then it is conducted the analysis of polymorphous loci of glutathione S-transferase M1 genes (GSTM1), N-acetyltransferase 2 (Nat2), angiotensin converting enzyme (ACE) and plasminogene activator (PA) due to the method of polymerase chain reaction of DNA synthesis (PCR) and at revealing genotype combinations of 0/0,S/R,I/D,I/D; +/+,R/R,I/I,I/D; +/+,S/S,I/D,I/D; +/+,S/R,I/I, I/D; +/+,S/R,D/D,I/I the risk of iron deficiency anemia development is predicted. EFFECT: higher accuracy of prediction. 2 cl, 2 ex, 1 tbl

Description

 The proposed group of inventions relates to medicine, namely to hematology, and can be used to predict the development of iron deficiency anemia (IDA).

 IDA is an urgent public health problem, which is explained by the widespread occurrence of sideropenia among all population groups, especially of working age, and an increase in recurrent, severe and resistant forms of the disease. This implies the need for large economic costs associated with significant labor losses, which determines the important socio-economic importance of the studied problem. Primary prevention of IDA is often carried out with existing latent iron deficiency. Therefore, the search for modern methods for predicting IDA is relevant.

 A known method for predicting the development of IDA by determining microelementosis (iron and copper deficiency) in soil and water [Nikitin E.N. Iron deficiency conditions in the population in the Middle Urals region: Abstract. dis .... d-pa honey. Sciences .- M., 2001.- 43 p.].

 The prototype is a method for predicting the development of IDA in identifying risk factors by interrogation (hyperpolymenorrhea, frequent pregnancies (4 or more) and childbirth (3 or more), prolonged lactation (more than 24 months), chronic bleeding, milk and vegetable diet) [Miterev Yu. G. Iron deficiency anemia: Achievements and problems // Hematol. and transfusiol.-1983.-N6.-C.3-8.], [Iron deficiency anemia / Ed. V.I. Nikulicheva. -Ufa, 1993.-201 p.].

 However, these methods for predicting the development of IDA are carried out against the background of a possible latent iron deficiency, are inaccurate and generalized.

 EFFECT: increased accuracy of forecasting in the absence of latent iron deficiency.

 The specified technical result is achieved by the fact that polymorphic loci of the cytochrome P-4501A1 gene (CYP1A1) are analyzed by the method of polymerase chain reaction of DNA synthesis (PCR) using the method of phenol-chloroform extraction of DNA from peripheral venous blood lymphocytes. After amplification and restriction, DNA fragments are separated electrophoretically in a 7% polyacrylamide non-denatured gel and analyzed under ultraviolet light on a transilluminator: the presence of DNA fragments of 48 and 139 nucleotide pairs (bp) is interpreted as an isoleucine (He) allele, 48, 120 and 19 bp - like valine. If the examined genotype Ile / Val is detected, the risk of developing IDA increases 2.5 times (option 1).

The method is as follows. Amplification of the polymorphic region of the CYP1A1 gene is carried out in a reaction mixture containing about 100 ng of genomic DNA; 1 unit Taq polymerase, 0.25 mg of the corresponding primers -5'-GAACTGCCACTTCAGCTG TST-3 'and 5'-GAAAGACCTCCCAGCGGTCA-3' 200 mM each dNTP in 50 ml of 1HPCR buffer. The amplification mode is as follows: initial denaturation at 95 ^o C for 10 min, 35 cycles, including: denaturation at 95 ^o C - 1 min, annealing of primers at 55 ^o C - 1 min, synthesis at 72 ^o C - 2 min. After amplification, the PCR products are hydrolyzed with a Hindl restriction endonuclease. For this, 10 μl of the PCR product is mixed with 5 units of the enzyme, the mixture is kept at 37 ^° C for 10 hours. Electrophoresis of DNA fragments is carried out in 1 x trisborate buffer (0.089 M Tris-Hcl pH 7.8; 0.089 M boric acid; 0.002 M EDTA with pH 8.0) in vertical plates at a constant voltage of 250-300 V after a 30-minute pre-electrophoresis. Before applying to the gel, the samples are mixed in a 1: 5 ratio with paint containing 0.25% bromophenol blue, 0.25% xylencyanol, 15% ficol. After electrophoresis, the gel is stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator.

A significant difference was found between the frequency distribution of the Ile / Val genotype of the CYP1A1 gene in IDA patients (N = 93) and the control group (N = 70): 16.12 and 5.71%, respectively (χ ² = 5.57; p = 0.044) . The calculation of the relative risk (OR) of the occurrence of IDA in the presence of the Ile / Val genotype of the CYP1A1 gene is performed according to the formula OR = a * d / b * c, where a is the genotype frequency in the patient sample (in%); b - genotype frequency in the control sample (in%); c = 100-a, d = 100-b, the risk of developing the disease is ascertained at OR> 1.5. In our study, OR = 2.54 in the presence of the Ile / Val genotype of the CYP1A1 gene. This genotype is a diagnostic marker for IDA and can be used to identify a risk group for a disease before detecting latent iron deficiency in order to conduct preventive courses of iron therapy.

 Example 1. Patient K-va R. X., 40 years old, has been under observation since 23. 11.1999, with severe severe iron deficiency anemia due to uterine blood loss. The patient was admitted with complaints of weakness, dizziness, pallor and dry skin, hair loss, brittle nails, palpitations, shortness of breath with little physical exertion. The patient considers himself 8 years old, has not received treatment in the past. She was born the third child in a row, mother and sisters suffered from anemia. Menstruation for 3 days, not abundant, cycle of 25 days, pregnancy - 2, childbirth - 2. Nutrition is balanced. An objective examination of the state of moderate severity, the skin is pale, with a yellowish tinge, dry. The nail plates are soft, brittle, koilonychia. Above the lungs, vesicular breathing, a heart rate of 80 per minute, blood pressure of 110 and 70 mm RT. Art. The abdomen is soft, painless; the liver and spleen are not enlarged. In a general blood test, severe hypochromic anemia with microcytosis and anisocytosis, in the study of ferrokinetics - a decrease in serum iron and ferritin. After treatment with ferroplex for 3 months, a positive dynamics of the disease was noted. To study the genotype of the CYP1A1 gene, 4 ml of venous blood was taken from the patient, followed by DNA extraction and amplification of the polymorphic region of the CYP1A1 gene in a reaction mixture containing about 100 ng of genomic DNA; 1 Taq polymerase unit, 0.25 mg of the corresponding primers - 5'-GAACTGCC ACTTCAGCTGTCT-3'- and 5'-GAAAGACCTCCCAGCGGTCA-3 ', 200 mM of each dNTP in 50 ml of 1HPCR buffer. After restriction of the amplified PCR products with HincII endonuclease, electrophoresis of the fragments was carried out at a constant voltage of 250-300 volts after a 30-minute pre-electrophoresis. After electrophoresis, the gel was stained with ethidium bromide solution for 10 min and analyzed under ultraviolet light on a transilluminator. A study of the patient's CYP1A1 gene revealed the Ile / Val genotype. Conducting a genetic analysis long before the onset of disease symptoms would allow timely preventive measures to prevent the development of IDA.

 Example 2. A female donor of 32 years old was examined before the next (second) blood donation. No complaints. She was born the first child in a row, the heredity by IDA is not burdened. Menstruation of 3 days, mild, cycle of 29 days, there was 1 pregnancy, which ended in childbirth. The nutrition is balanced. An objective examination of the condition is satisfactory, no physical changes were detected. In the general analysis of blood, no abnormalities were found, serum iron and ferritin are within normal limits. To study the genotype of the CYP1A1 gene, 4 ml of venous blood was taken from the examined patient, followed by DNA extraction and amplification of the polymorphic region of the CYP1A1 gene in a reaction mixture containing about 100 ng of genomic DNA; 1 Taq polymerase unit, 0.25 mg of the corresponding primers - 5'-GAACTGCCACTTCAGCTGTCT-3 'and 5'-GAAAGACCTCCCAGCGG TCA-3', 200 mM each dNTP in 50 ml of 1HPCR buffer. After restriction of the amplified PCR products with HincII endonuclease, electrophoresis of the fragments was carried out at a constant voltage of 250-300 V after a 30-minute pre-electrophoresis. After electrophoresis, the gel was stained with ethidium bromide solution for 10 min and analyzed under ultraviolet light on a transilluminator. A study of the examined CYP1A1 gene revealed the Ile / Val genotype. Conclusion: it is necessary to conduct preventive courses of treatment with iron preparations to prevent the development of IDA, since the presence of this genopip increases the risk of developing the disease 2.5 times.

The prototype of option 2 of the present invention is a method for predicting the development of IDA in identifying risk factors by interrogation (hyperpolymenorrhea, frequent pregnancies (4 or more) and childbirth (3 or more), prolonged lactation (more than 24 months), chronic bleeding, dairy-vegetable diet) [Miterev Yu.G. Iron deficiency anemia: Achievements and problems // Hematol. and transfusiol.-1983.-N6.-C.3-8.]. [Iron deficiency anemia / Ed. V.I. Nikulicheva. Ufa, 1993.-201 p.]
However, this method for predicting the development of IDA is carried out against the background of a possible latent iron deficiency, is inaccurate and generalized.

 EFFECT: increased accuracy of forecasting in the absence of latent iron deficiency.

 The indicated technical result is achieved by the analysis of polymorphic loci of the genes directly or indirectly involved in the IDA pathogen selected in the method of phenol-chloroform extraction of DNA from peripheral venous blood lymphocytes: glutathione S-transferase Ml (GSTM1), N-acetyltransferase 2 (Nat2) , angiotensin-converting enzyme (ACE) and plasminogen activator (AP) by polymerase chain reaction of DNA synthesis (PCR). After amplification and restriction, the DNA fragments are separated electrophoretically in a 7% polyacrylamide non-denatured gel and analyzed under ultraviolet light on a transilluminator. The presence of homo- and heterozygotes according to the normal allele of the GSTM1 gene (genotypes + / + and + / 0, respectively) is determined by the presence of an amplification fragment of 271 bp on electrophoregrams; its absence (0/0) indicates a homozygous state for deletion of this gene region. The presence of a 547 bp Nat2 gene fragment containing three polymorphic sites detected by the restriction enzymes Kpnl, TagI, and BamH is interpreted as a fast form of the Nat2 gene (genotype R / R); the loss of one of the restriction sites leads to the appearance of a mutant allele, which results in the genotype S / S or S / R of the Nat2 gene. The polymorphism of the ACE and AP genes is due to the presence (insertion) or absence (deletion) of the Alu repeat, resulting in the visualization of 490 bp alleles. - insertion (I) or 190 bp - divisions (D), respectively. In accordance with the presence of insertion / deletion in the homo- or heterozygous state, the following genotypes are formed: I / I - homozygous for the presence of insertion; D / D - deletion homozygote; I / D - heterozygote.

 If the examined combinations of genotypes are shown in table 1, the risk of developing IDA increases by 3.8-20.4 times (option 2).

The method is as follows. Amplification of the GSTM1 gene fragment is carried out in a reaction mixture containing approximately 100 ng of genomic DNA, 15 mM each primer (5'-GAACTCCCTGAAAAGCTAAAGC-3 'and 5'-GTTGGGCTCAAATATACGGTGG-3'), 67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 6.7 mM MgCl ₂ , 6.7 μM EDTA, 10 mm mercaptoethanol, 170 μg BSA, 1.0 mm each dNTP and 1 unit of Taq polymerase. The amplification mode is as follows: initial denaturation (94 ^o C, 7 min), 30 cycles of amplification: denaturation -94 ^o C, 40 s; annealing - 55 ^o С, 40 s, synthesis - 72 ^o С, 1 min. In the absence of a deletion, a 400 bp fragment is formed; in the presence of a deletion, this fragment is absent. As an internal control, amplification of the CYP1A1 gene fragment is used.
Amplification of the Nat2 gene fragment is performed in 25 μl of the total volume of the mixture containing 67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 16.7 mM MgCl ₂ , 6.7 μM EDTA, 10 mM mercaptoethanol, 170 μg BSA 1.0 mM of each dNTP, 1 unit of Taq polymerase and the corresponding primers of 15 nM each (5 -GCTGGGTCTGGAAGCTCCTC-3 'and 5'-TTGGGTGATACATAGACAAG GG-3'). After denaturation (94 ^o C, 7 min), 30 amplification cycles are carried out in the mode: 94 ^o C - 40 s, 59 ^o C - 40 s, 72 ^o C - 1 min with final synthesis for 5 min at 72 ^o C. At to identify the mutant allele of the Nat2 gene, the resulting PCR product is digested with restriction enzymes Kpnl, Taql, BamHl.

Amplification of the polymorphic portion of the ACE gene is carried out in 20 μl of the reaction mixture containing 50 mm Tris-HCl, pH 8.8; 15 mM (NH ₄ ) ₂ SO ₄ ; 5 mM MgCl ₂ ; 0.01% Tween-20; 0.01% gelatin; 0.1 μg of genomic DNA; dNTP mixture (200 μg each) and corresponding primers of 10 pM each (5'-CTGGAGACCACTCCCATCCTTTCT-3 'and 5'-GATGTGGCCATCACATTCGTC AGAT-3'). After denaturation at 95 ^{° C.} for 1 minute, 1 unit of Taq polymerase is added. Subsequent cycles include: denaturation - 1 min at 94 ^° C, annealing - 1 min at 62 ^° C, elongation - 1 min at 72 ^° C. After 30 cycles of amplification, the samples are held for 10 min at 72 ^° C and cooled.

Amplification of the polymorphic region in the AP gene is carried out in 25 μl of the reaction mixture of the following composition: 50 mM Tris-HCl, pH 8.8; 15 mM (NH ₄ ) ₂ SO ₄ ; 5 mM MgCl ₂ ; 0.01% Tween-20; 0.01% gelatin; 0.1 μg of genomic DNA; dNTP mixture (200 μg each) and corresponding primers of 10 pM each (5'-GTAAGAGTTCCGTAACAGGACAGCT-3 'and 5'-CCACCCTAGG AGAACTTCTCTCTTT-3'). The parameters of 30 amplification cycles are as follows: denaturation - 1 min at 94 ^o C, annealing - 1 min at 58 ^o C, elongation - 1 min at 72 ^o C.

 Amplified DNA fragments are separated electrophoretically in a 7% polyacrylamide non-denatured gel. Electrophoresis of DNA fragments is carried out in 1 trisborate buffer (0.089 M Tris-HCl pH 7.8; 0.089 M boric acid; 0.002 M EDTA with pH 8.0) in vertical plates at a constant voltage of 250-300 V after a 30-minute pre-electrophoresis. Before applying to the gel, the samples are mixed in a 1: 5 ratio with paint containing 0.25% bromophenol blue, 0.25% xylencyanol, 15% ficol. After electrophoresis, the gel is stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator.

 A significant difference was found between the frequency distribution of combinations of genotypes of the GSTM1, Nat2, ACE and AP genes in IDA patients (N = 99) and the control group (N = 90) (p <0.05). The calculation of the relative risk (OR) of the occurrence of IDA in the presence of combinations of genotypes shown in the table is carried out according to the formula OR = a * d / b * c, where a is the frequency of combinations of genotypes in the patient sample (in%); b is the frequency of combinations of genotypes in the control sample (in%); c = 100-a, d = 100-b, the risk of developing the disease is ascertained at OR> 1.5. In our study, OR - from 3.83 to 20.4 in the presence of combinations of genotypes presented in the table. These combinations of genotypes are diagnostic markers of IDA and can be used to identify a risk group for developing the disease before detecting latent iron deficiency during screening studies or genetic passportization of the population in order to conduct preventive courses of iron therapy.

Example 1. Patient A-va L. T., 43 years old, has been under observation since 3.12.1999 for severe iron deficiency anemia due to uterine blood loss. The patient was admitted with complaints of weakness, dizziness, pallor and dry skin, hair loss, brittle nails, palpitations, shortness of breath with little physical exertion. The patient considers himself 12 years old, has not received treatment in the past. She was born the 2nd child, mother and sisters suffered from anemia. Menstruation for 7 days, heavy, regular, cycle 28 days, pregnancy - 4, childbirth - 3, abortion - 1. Nutrition is balanced. An objective examination of the state of moderate severity, the skin is pale, dry. The nail plates are soft, brittle, koilonychia. Vesicular respiration over the lungs, heart rate 90 per minute, blood pressure 100 and 65 mm RT. Art. The abdomen is soft, painless; the liver and spleen are not enlarged. In a general blood test, severe hypochromic anemia with microcytosis and anisocytosis, in the study of ferrokinetics - a decrease in serum iron and ferritin. After treatment with ferroplex for 3 months, no positive dynamics of the disease were noted. To study the combinations of genotypes of the GSTM1, Nat2, ACE, and AP genes, 4 ml of venous blood was taken from the patient, followed by DNA extraction and amplification of polymorphic sections of the GSTM1, Nat2, ACE and AP genes in reaction mixtures containing, respectively, for the GSTM1 gene: 100 ng of genomic DNA , 15 pM of each primer (5'-GAACTCCCTGAAAAGCTAAA GC-3 'and 5 - GTTGGGCTCAAATATACGGTGG-3'), 67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 6.7 mM MgCl ₂ , 6.7 μM EDTA, 10 mM mercaptoethanol, 170 μg BSA, 1.0 mM each dNTP and 1 unit of Taq polymerase; for the Nat2 gene: 67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 16.7 mM MgCl ₂ , 6.7 μM EDTA, 10 mm mercaptoethanol, 170 μg BSA, 1.0 mm each dNTP, 1 units of Taq polymerase and corresponding primers of 15 nM each (5'-GCTGGGTCTGGAAGCTCCTC-3 'and 5 - TTGGGTGATACAT AGACAAGGG-3'), followed by restriction of the PCR products of the Nat2 gene with restriction enzymes Kpnl, Taql, BamHl; for ACE gene: 50 mM Tris-HCl, pH 8.8; 15 mM (NH ₄ ) ₂ SO ₄ ; 5 mM MgCl ₂ 0.01% Tween-20; 0.01% gelatin; 0.1 μg of genomic DNA; dNTP mixture (200 μg each) and corresponding primers of 10 pM each (5'-CTGGAGACCACTCCCATCCTTTCT-3 'and 5'-GATGTGGCCATCA CATTCGTCAGAT-3'); for the AP gene: 50 mM Tris-HCl, pH 8.8; 15 mM (NH ₄ ) ₂ SO ₄ ; 5 mM MgCl ₂ ; 0.01% Tween-20; 0.01% gelatin; 0.1 μg of genomic DNA; dNTP mixture (200 μg each) and corresponding primers of 10 pM each (5'-GTAAGAGTTCCGTAACAGGACAGCT-3 'and 5 - CCACCCTAGGAGAACTTCT CTTT-3'). Amplified DNA fragments were separated electrophoretically in a 7% polyacrylamide undenatured gel under a voltage of 250-300 V after a 30-minute pre-electrophoresis. After electrophoresis, the gel was stained with ethidium bromide solution for 10 minutes and analyzed under ultraviolet light on a transilluminator. The study of gene combinations of GSTM1, Nat2, ACE and AP genes revealed a combination of + / +, S / S, I / D, I / D genotypes. Conducting a genetic analysis long before the onset of disease symptoms would allow timely preventive measures to prevent the development of a severe, resistant form of IDA.

Example 2. A female donor, 25 years old, was examined before the first blood donation. No complaints. She was born the first child in a row, the heredity by IDA is not burdened. Menstruation for 4 days, mild, cycle 28 days, there were no pregnancies. The nutrition is balanced. An objective examination of the condition is satisfactory, no physical changes were detected. In the general analysis of blood, no abnormalities were found, serum iron and ferritin are within normal limits. To study the combinations of genotypes of the GSTM1, Nat2, ACE, and AP genes, 4 ml of venous blood was taken from the patient, followed by DNA extraction and amplification of polymorphic sections of the GSTM1, Nat2, ACE and AP genes in reaction mixtures containing, respectively, for the GSTM1 gene: 100 ng of genomic DNA , 15 pM of each primer (5'-GAACTCCCTGAAAAGCTAAAGC-3 'and 5'-GTTGGGCTCAAATATACGGTGG-3'), 67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 6.7 mM MgCl ₂ , 6 , 7 μM EDTA, 10 mM mercaptoethanol, 170 μg BSA, 1.0 mM each dNTP and 1 unit of Taq polymerase; for the Nat2 gene: 67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 16.7 mM MgCl ₂ , 6.7 μM EDTA, 10 mm mercaptoethanol, 170 μg BSA, 1.0 mm each dNTP, 1 u Taq polymerase and corresponding primers of 15 nM each (5'-GCTGGGTCTGGAAGCTCCTC-3- and 5'-TTGGGTGATACATAG ACAAGGG-3 ') followed by restriction of the PCR products of the Nat2 gene with Kpnl, Taql, BamHl; for ACE gene: 50 mM Tris-HCl, pH 8.8; 15 mM (NH ₄ ) ₂ SO ₄ ; 5 mM MgCl ₂ 0.01% Tween-20; 0.01% gelatin; 0.1 μg of genomic DNA; dNTP mixture (200 μg each) and corresponding primers of 10 pM each (5-CTGGAGACCACTCCCATCCTTTCT-3- and 5'-GATGTGGCCATCA CATTCGTCAGAT-3 '); for the AP gene: 50 mM Tris-HCl, pH 8.8; 15 mM (NH ₄ ) ₂ SO ₄ ; 5 mM MgCl ₂ ; 0.01% Tween-20; 0.01% gelatin; 0.1 μg of genomic DNA; dNTP mixture (200 μg each) and corresponding primers of 10 pM each (5'-GTAAGAGTTCCGTAACAGGACAGCT-3 'and 5'-CCACCCTAGGAGAACTTC TSTTT-3'). Amplified DNA fragments were separated electrophoretically in a 7% polyacrylamide undenatured gel under a voltage of 250-300 V after a 30-minute pre-electrophoresis. After electrophoresis, the gel was stained with ethidium bromide solution for 10 min and analyzed under ultraviolet light on a transilluminator. The study of gene combinations of the genes GSTM1, Nat2, ACE and AP revealed a combination of genotypes + / +, S / R, D / D, I / I. Conclusion: it is necessary to conduct preventive courses of treatment with iron preparations for 7-10 days after menstruation to prevent the development of IDA and removal from donation, since the presence of this genopip increases the risk of developing the disease by 12.8 times.

 The proposed method is modern and allows a high degree of certainty to predict the disease.

Claims (2)

 1. A method for predicting the development of iron deficiency anemia, characterized in that DNA is isolated from peripheral venous blood lymphocytes by the polymerase chain reaction of DNA synthesis, fragments of the cytochrome P-4501 A1 gene are amplified, and if the isoleucine / valine genotype is detected, the risk of developing iron deficiency anemia in the subject is predicted.  2. A method for predicting the development of iron deficiency anemia, characterized in that DNA is isolated from peripheral venous blood lymphocytes by the polymerase chain reaction of DNA synthesis, gene fragments of glutathione S-transferase M1, N-acetyltransferase 2, angiotensin-converting enzyme and plasminogen activator are amplified and the identification of combination 0/0, S / R, I / D, I / D; + / +, R / R, I / I, I / D; + / +, S / S, I / D, I / D; + / +, S / R, I / I, I / D; + / +, S / R, D / D, I / I predict the risk of developing iron deficiency anemia in the subject.

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