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Table of Contents   
ORIGINAL ARTICLE
Year : 2022  |  Volume : 27  |  Issue : 6  |  Page : 699-706
 

MTHFR C677T and MTR A2756G gene polymorphism in neural tube defect patients and its association with red blood cell folate level in Eastern Indian Population


1 Department of Biochemistry, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
2 Department of Pediatric Surgery, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
3 Department of Reproductive Medicine, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
4 Department of Biochemistry, Netaji Subhas Medical College and Hospital, Dayalpur Daulatpur, Bihar, India

Date of Submission12-Feb-2022
Date of Decision14-May-2022
Date of Acceptance27-Jun-2022
Date of Web Publication11-Nov-2022

Correspondence Address:
Vinit Kumar Thakur
Department of Pediatric Surgery, Indira Gandhi Institute of Medical Sciences, Patna - 800 014, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaps.jiaps_29_22

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   Abstract 


Introduction: Single-nucleotide polymorphism (SNP) is a single-nucleotide change in a deoxyribose nucleic acid (DNA) sequence that occurs in >1% of population. Methylene tetra hydro folate reductase (MTHFR) C677T (rs1801133) and methionine synthase enzyme (MTR) A2756G (rs1805087) are two such SNPs occurring in coding sequence of the respective genes, which are frequently seen with neural tube defects (NTDs). MTHFR and MTR genes are involved in folate metabolism. The folate level in the course of pregnancy is treated as vital in the etiopathogenesis of NTDs. This study aims to explore the association of SNPs of both genes and red blood cell (RBC) folate levels in the predisposition to NTDs.
Aims and Objective: The purpose of this investigation was to determine the relationship of NTDs with polymorphisms in MTHFR and MTR genotype and to estimate and compare the RBC folate levels in NTD patients and controls.
Materials and Methods: A total of 397 individuals were enrolled (163 patients and 234 controls) for this observational study. Genotyping to find out MTHFR C677T and MTR A2756G was performed by polymerase chain reaction–restriction fragment length polymorphism technique from DNA extracted from the subject's blood. RBC folate level was estimated by chemiluminescence immunoassay method with the same blood sample.
Results: The total RBC folate levels were significantly less among cases compared to controls (P = 0.020). A significant difference for RBC folate was observed between case and control groups of various genotypes of MTHFR C677T, except heterozygote CT (P = 0.459). Among MTR A2756G, genotypes with only homozygous AA have significant difference (P = 0.003) for RBC folate levels. Among different types of NTDs, there were no significant differences for RBC folate levels. Among MTHFR C677T, T allele possessed 1.9 times risk compared to C allele for the occurrence of NTDs. In MTR A2756G polymorphism, the odds of developing NTDs were 1.6 times in heterozygous AG compared to homozygous AA. Similarly, the risk for NTDs was three times higher in subjects with both heterozygous AG and CT genotypes compared to wild-type homozygous AA and CC genotypes.
Conclusion: The total RBC folate levels were significantly less among cases compared to controls, and the genotypes had no such effect in decrease in RBC folate levels. The presence of mutant allele in homozygous or heterozygous condition for both SNPs had increased risk associated with NTDs.


Keywords: Methylene tetra hydro folate reductase C677T, methionine synthase A2756G, neural tube defects, red blood cell folate, single-nucleotide polymorphism


How to cite this article:
Kumari R, Kumar S, Thakur VK, Singh K, Kumar U. MTHFR C677T and MTR A2756G gene polymorphism in neural tube defect patients and its association with red blood cell folate level in Eastern Indian Population. J Indian Assoc Pediatr Surg 2022;27:699-706

How to cite this URL:
Kumari R, Kumar S, Thakur VK, Singh K, Kumar U. MTHFR C677T and MTR A2756G gene polymorphism in neural tube defect patients and its association with red blood cell folate level in Eastern Indian Population. J Indian Assoc Pediatr Surg [serial online] 2022 [cited 2022 Nov 30];27:699-706. Available from: https://www.jiaps.com/text.asp?2022/27/6/699/360956





   Introduction Top


The enzyme methylene tetra hydro folate reductase (MTHFR) is the key regulator of folate metabolic pathway and regulates intracellular folate concentration for the synthesis and methylation of deoxyribose nucleic acid (DNA). The MTHFR gene is situated on the short arm of chromosome 1 and has 11 exons. At nucleotide 677 in the 4th exon, there is substitution of cytosine (C) by thymine (T) nucleotide, resulting in an amino acid substitution of alanine to valine.[1] This change decreases the enzyme activity, resulting in decreased folate levels in the red blood cells (RBCs), plasma, and serum, while increased total homocysteine levels in the plasma.[2] Methionine synthase (MTR), located on long arm of chromosome 1, is one of the core factors in the folate pathway. The MTR gene codes for a protein of 1265 amino acids which functions as a key enzyme in DNA repair, synthesis, and methylation. The A2756G single-nucleotide polymorphism (SNP) or rs1805087 in MTR gene coding sequence results in change of nucleotide A to G that leads to substitution of aspartic acid to glycine residue at position 919 (D919G) in the peptide sequence.[3]

A2756G SNP has been demonstrated to contribute to change in levels of homocysteine and folate in the plasma.[4] Low serum folate levels are associated with increased risk of neural tube defects (NTDs). RBC folate level can also be measured to assess the risk of NTDs.[5] NTDs are congenital anomalies of the central nervous system occurring due to improper closure of the neural tube. The prevalence of NTDs in India has been reported to vary from 0.5 to 11 per 1000 live births.[6],[7],[8],[9] Some studies have reported the prevalence of myelomeningocele (MMC) in northern India to be 3.15 per 1000 live births.[7] The MMCs may occur in any part of the spine, but it is predominately observed in the lumbosacral region, the last region of the neural tube to fuse, with thoracolumbar lesions being less typical.[10] The thermolabile MTHFR C677T (A222V) variant is a risk factor for NTDs in some but not all populations and is associated with low serum and RBC folate levels and elevated homocysteine levels.[11] Diminished level of folate and Vitamin B12 and high level of homocysteine in the serum during pregnancy are well known to increase the risk of NTDs.[12] Previous studies have reported the association of MTHFR C677T and MTR A2756G gene with NTDs.[13],[14] We have made an attempt to determine the risk associated with MTHFR (C677T) and MTR (A2756G) gene polymorphism as well as the level of RBC folate in patients with NTDs.


   Materials and Methods Top


Study design

A prospective observational study of children born with NTD was done on patients attending the pediatric surgery department in a tertiary care center in a period of 2½ years. The study population comprised 163 NTD cases (male = 105; female = 58) and 234 healthy controls (male = 124, female = 109) taken from normal population of the same age group. The subjects were not related and belonged to the eastern part of India. Each study participant was made aware of the nature and scope of the study. Data and blood collection from all individuals were taken after having informed written consent/assent from their parents.

Inclusion criteria

  1. NTD cases of 0–14 years of age and both sexes visiting the Department of Pediatric Surgery, Indira Gandhi Institute of Medical Sciences (IGIMS), Patna.


Exclusion criteria

  1. Not ready to participate
  2. Other major health problems in which mother or child suffering from disease such as cancer, tuberculosis, and epilepsy requiring medication that can affect RBC folate level
  3. Mother on medication such as oral contraceptives
  4. Mother or child on anticonvulsants and antiepileptic.


Red blood cell folate level estimation

A volume of 0.5 ml of peripheral blood was collected in Ethylenediaminetetraacetic acid (EDTA) microtainers from each individual; 50 μl of blood was pipetted into 1 ml of RBC lysing agent (Beckman Coulter, USA) and mixed well. The solution was allowed to stand in dark for 90 min, and then, the hemolysate was run on automated chemiluminescence immunoassay analyzer (Access 2, Beckman and Coulter, USA) for folate estimation. The result of RBC folate level was calculated from hemolysate folate value by the following formula:



Deoxyribose nucleic acid isolation and genotyping

Genomic DNA was isolated from 200 μl peripheral blood collected in the EDTA microtainer using Qiagen Spin column method (Qiagen, Germany) according to the manufacturer's instructions. Quantification of extracted DNA was done using NanoDrop spectrophotometer. The average A260/280 and A260/230 ratio of DNA was 1.8 and 2.1, respectively. The average concentration of DNA extracted was 28 ng/μl. The location and ID of polymorphism were confirmed with the National Center for Biotechnology Information SNP database. The MTHFR C677T (rs1801133) and MTR A2756G (rs1805087) gene polymorphisms were determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism genotyping technique. The primers for the above SNP sites were made and verified by Primer BLAST online software (http://www.ncbi.nlm.nih.gov/tools/primer-blast). The MTHFR C677T polymorphism was detected by target DNA amplification using the forward primer 5'-TGA AGG AGA AGGTGT CTGCGGGA-3' and the reverse primer 5'-AGG ACGGTG CGG TGA GAG TG-3' described by Matsuo et al.[15] For the detection of genotypes of MTR A2756G variants, following site-specific oligonucleotide primers were used: forward primer 5'-TGT TCC AGA CAG TTA GAT GAA AAT C-3' and reverse primer 5'-GAT CCA AAG CCT TTT ACA CTC CTC-3'.

Polymerase chain reaction conditions

PCR for both MTHFR and MTR genes was done on VeritiPro thermal cycler (Applied Biosystems, USA) using 50 μl of final reaction mixture containing 5 μl DNA (50–100 ng of genomic DNA), 25 μl PCR mix (AmpliTaq Gold 360, Applied Biosystems, USA), 1 μl each primer (100 pmol/ml), and 18 μl sterile distilled water to make up the final volume. PCR thermal cycling conditions for MTHFR C677T were initial denaturation for 8 min at 95°C, followed by 40 cycles of denaturation for 45 s at 95°C, annealing for 1 min at 62°C, extension for 40 s at 72°C, and one cycle final extension for 7 min at 72°C. For MTR gene, the conditions were as follows: initial denaturation for 8 min at 95°C, followed by 35 cycles with denaturation for 1 min at 95°C, annealing for 1.5 min at 61°C, extension for 1 min at 72°C, and one cycle of final extension for 7 min at 72°C. The amplified PCR products were 198 bp for MTHFR gene and 211 bp for MTR gene.

Restriction fragment length polymorphism conditions

The restriction digestion was performed by using 2 μl of Hinfl restriction enzyme (New England Biolabs, USA), 2 μl NEB restriction buffer, 15 μl of amplified MTHFR C677T PCR product, and 1 μl of sterile distilled water to reach total volume of 20 μl, and the same volumes were used for MTR A2756G PCR product with restriction enzyme Hae III (New England Biolabs, USA). The restriction digest was incubated for 4 h at 37°C in both the cases.

Detection of genotypes

The products of restriction digestion were separated on a 4% agarose gel prestained with ethidium bromide and analyzed under ultraviolet transilluminator. For MTHFR C677T polymorphism, the wild-type genotype CC showed only one undigested band of 198 bp; heterozygous CT genotype was identified by the presence of 3 bands of 198 bp, 175 bp, and 23 bp. Homozygous TT mutant genotype was identified by the presence of two bands of 175 bp and 23 bp. The 23 bp band here was not visible properly on the gel due to lack of sensitivity of ethidium and background staining noise [Figure 1]. For MTR A2756G polymorphism, the wild-type genotype AA showed only one undigested band of 211 bp; heterozygous AG genotype was identified by the presence of 3 bands of 211 bp, 131 bp, and 80 bp. Homozygous GG mutant genotype was identified by the presence of two bands of 131 bp and 80 bp [Figure 2].
Figure 1: 4% agarose gel showing restriction digest products of MTHFR PCR product. Lane 1 is 50 bp DNA ladder, lane 2, 3, 4, and 9 heterozygous, and lane 5, 6, 7, 8 and 10 wild-type homozygous, MTHFR: Methylene tetra hydro folate reductase, PCR: Polymerase chain reaction, DNA: Deoxyribose nucleic acid

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Figure 2: 4% agarose gel showing restriction digest products of MTR PCR product. From left to right, lane 1 is 50 bp DNA ladder, lane 5 and 7 heterozygous, and lane 2, 3, 4, 6, and 8 wild-type homozygous, MTR: Methionine synthase, PCR: Polymerase chain reaction, DNA: Deoxyribose nucleic acid

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Statistical analysis

Data were presented as mean ± standard deviation and percentages. Mann–Whitney U-test and Kruskal–Wallis test were performed to calculate the difference between the patients, controls group, and genotype subgroups. Binary logistic regression was used to evaluate the maternal risk factor associated with NTDs. For the variables to be included in the logistic regression model, we used a forward likelihood ratio selection with a P = 0.05 for the addition of variables and P = 0.10 for the removal. The allele frequencies were calculated by allele counting for each genetic marker. Hardy–Weinberg equation and the differences in genotypic frequencies were examined using online calculator Gene-calc (https://gene-calc.pl). The odd ratios (ORs) for different MTHFR and MTR genotypes/alleles were calculated with 95% confidence interval (CI) by Chi-square test, and concurrently, P values were calculated to evaluate the association with NTDs. P < 0.05 was considered statistically significant. All statistical analyses were done using Statistical Package for the Social Sciences (SPSS) software version 16 (IBM, USA).


   Results Top


The present study comprised 163 cases of NTD and 234 healthy control individuals. There were 105 (64.4%) male and 58 (35.6%) female NTD patients, while there were 124 (53.2%) and 109 (46.8%) male and females in the control group. There was a significant difference in the distribution of sex between case and control groups (χ2 = 4.932, df = 1, P = 0.026). The total RBC folate levels were significantly less among cases compared to controls (P = 0.020). Similarly, the total RBC folate levels were significantly less among cases compared to controls for various genotypes of MTHFR C677T, except heterozygous CT (P = 0.459). It was observed in MTR A2756G genotypes that only homozygous AA had significant difference (P = 0.003) of RBC folate levels between case and control groups [Table 1].
Table 1: Comparison of red blood cell folate levels between cases and control according to sex or genotypes

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Most of the cases comprised lumbosacral type of NTD (n = 122 [74.8%]) followed by sacral type (n = 21 [12.9%]) and the rest were thoracic and cervical NTD with n = 12 (7.4%) and n = 8 (4.9%) cases, respectively. There was no significant difference for RBC folate levels among different types of NTD [Table 2]. The most cases of NTDs associated with CC genotype of MTHFR C677T (n = 80 [49.1%]) and with AA genotype of MTR A2756G were lumbosacral MMC (n = 59 [36.2%]) [Table 3].
Table 2: Comparison of red blood cell folate levels among different types of neural tube defects

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Table 3: Distribution of types of neural tube defect according to genotypes

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Binary logistic regression results indicated maternal risk factors such as not taking folate supplements during early pregnancy and age >35 years. However, parity, maternal body mass index (BMI), and income status were not associated with the risk of NTDs [Table 4]. Familial income was significantly different between NTDs and control group [Table 5]. The mothers with age more than 35 years had 2.7 times risk of NTD babies. Similarly, the mothers who had not taken folate supplement in the early pregnancy had 1.97 times risk of developing NTD babies. Maternal BMI whether underweight or overweight had no effect on susceptibility to NTDs [Table 6].
Table 4: The result of logistic regression analysis of maternal risk factor on neural tube defect

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Table 5: Comparison of maternal risk factors in neural tube defects and control group

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Table 6: Association of maternal risk factors and neural tube defects

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Distribution of genotypes was in accordance with Hardy–Weinberg equilibrium. The frequencies of MTHFR C677T CT versus CC differ significantly (OR = 1.8, 95% CI [1.1–2.9], P = 0.013). Also combined CT + TT versus CC model were different between groups (OR = 1.9, 95% CI [1.2–2.9], P = 0.007). Frequencies of T versus C were also significantly different (OR = 1.9, 95% CI [1.2–2.8], P = 0.003). In MTR A2756G polymorphism, the odds of developing NTDs were 1.6 times higher in heterozygote AG compared to homozygous AA (OR = 1.6, 95% CI [1.0–2.4], P = 0.029). There was no significant difference between frequencies of GG versus AA, AG + GG versus AA model, and G allele versus A allele [Table 7].
Table 7: Genotype and allele distribution of methylene tetra hydro folate reductase (C677T) and methionine synthase (A2756G) polymorphism among case and control groups

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For combined polymorphism of MTHFR C677T and MTR A2756G, following were the observations. There was no significant difference in frequencies for heterozygote CT of MTHFR C677T combination with homozygous AA of MTR A2756G compared to homozygous CC and AA genotypes among the groups. In addition, there was no significant difference when homozygous CC and heterozygous AG were compared to homozygous CC and AA (P = 0.101). The risk for NTDs was three times higher in subjects with both heterozygous AG and CT genotypes compared to wild-type homozygous AA and CC genotypes.(OR = 3.0, 95% CI [1.3–6.5], P = 0.006). Although there was no difference in frequencies of distribution of combined genotype homozygous CC and GG compared to wild-type homozygous CC and AA genotypes among cases and control group (P = 0.054) [Table 8].
Table 8: Combined genotype frequencies of methylene tetra hydro folate reductase C677T and methionine synthase A2756G gene polymorphism among case and control group

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   Discussion Top


There are many studies on genetic polymorphism of folate metabolism-related genes and risk of NTD as well as levels of folate in such patients. This study measures the levels of folate and genotypic frequencies of two such SNPs associated with folate metabolism in NTD patients to assess any associated risk, interaction, or possible correlation with this congenital anomaly.

RBC folate is a better indicator of folate status than serum folate because it reflects levels of intracellular folate and folate turnover during the past 120 days.[16] Nutritional and genetic factors may affect RBC folate status. Many studies have suggested that there is no significant decrease in folate levels in NTDs,[17],[18],[19],[20],[21],[22],[23] and equivocally, most have found lower serum folate level and RBC folate level[24],[25],[26],[27],[28] or amniotic fluid folate level[29] in mother carrying NTD fetus. Although there are many studies on maternal serum and maternal RBC folate level reporting their association with NTD, only a few are done on association of NTDs with child's RBC folate level. One such study was in agreement with the present study suggesting a significant decrease in RBC folate levels in NTDs compared to the controls.[25] There was clear indication in the present study that the RBC folate level was significantly decreased in NTD patients. However, when the RBC folate levels were compared in different types of NTDs, there was no significant difference between individual types of NTDs. This suggests that low RBC folate levels are equally associated with the various types of NTDs. This study also shows that there is decrease in folate levels in both sexes in NTD cases compared to control group, but there was no significant difference in RBC folate levels between sexes in either of the study groups. When RBC folate level was seen in MTHFR C677T genotypes, the total RBC folate levels were significantly less among cases compared to controls for various genotypes of MTHFR C677T, except heterozygous CT. Significant difference was observed in MTR A2756G homozygous AA genotype RBC folate levels between case and control groups [Table 1]. Similar results have been reported for MTHFR C677T polymorphism studies claiming heterozygous CT for the polymorphism which has 65% of the normal enzyme activity and 10% decreased RBC folate level; patients with the homozygous variant (TT) had only 30% of normal enzyme activity and 18% decreased RBC folate level.[30],[31] No study reports SNP A2756G of MTR gene influence on RBC folate levels in NTD subjects. There are only few studies reporting SNP A2756G of MTR gene with high incidence and were supposed to result in amino acid change which would further vary the function of folate-related enzymes.[24]

Overall, the total RBC folate levels were significantly less among cases compared to controls in the present study. However, none of the published researches have mentioned association and interaction of type of NTDs and folate levels.

There was no significant difference between different locations of NTDs and their respective RBC folate levels [Table 2]. Among both genotypes, lumbosacral MMC was the most common [Table 3].

NTDs have a multifactorial etiology including maternal, genetic, and environmental factors. Maternal factors can contribute to the development of NTDs. In this study, it was found that age >35 years at the time of delivery had 2.7 times risk for NTDs and the mothers who had not taken folate supplements had twofold risk of NTDs. BMI, parity, and income status had no effect on the occurrence of NTDs. A few studies have supported the opinion that genetic factors mainly involve the planar cell polarity signaling pathway,[32],[33] folate metabolism pathway,[34] and carbohydrate metabolism pathway.[35] However, the exact mechanism is yet not understood clearly.

There are many SNPs that have been shown to be associated with NTDs such as MTHFR C677T and MTR A2756G. The MTHFR C677T polymorphism with genotype heterozygous CT or TT has been reported to carry increased risk for NTDs formation in fetuses and newborns.[25] Similar observation was seen in the present study with frequencies of MTHFR C677T heterozygous CT versus CC had significant difference and there was 1.8 folds high risk for NTDs among heterozygous CT. Also combined dominant model possessed 1.9 times higher risk compared to wild-type CC homozygous. Risk associated to T allele was also 1.9 times more in comparison to C allele. In MTR A2756G polymorphism, the odds of developing NTDs were 1.6 times higher in heterozygous AG compared to homozygous AA. However, we did not observe significant difference between frequencies of GG versus AA and AG + GG versus AA model [Table 5]. Similar results were seen with 1.4 times high risk associated with G allele in NTDs compared to A allele.[36] To see the possible interaction of two genes and their possible association with NTDs, combined polymorphism of MTHFR C677T and MTR A2756G was analyzed. There was no significant difference in frequencies of individuals with heterozygous CT for MTHFR C677T and AA homozygous for MTR A2756G compared to individuals with homozygous CC and AA genotypes among the groups. The risk for NTDs was three times higher in subjects with both heterozygous AG and CT genotype compared to wild-type homozygous AA and CC genotypes. Further, there were no differences in frequencies of distribution of combined genotype homozygous CC and GG compared to reference genotypes.

All these combined effects of genotype shows that heterozygosity and mutant homozygosity for any of these SNPs are important risk factors for NTDs.


   Conclusion Top


The total RBC folate levels were significantly less among cases compared to controls and genotype had no such effect on decrease in RBC folate level. The presence of mutant allele in homozygous or heterozygous condition for both SNPs had increased risk associated with NTDs. Although this study cannot explain the exact mechanism, it is similar to most of the studies which had shown that SNP of MTHFR C677T (rs1801133) and MTR A2756G (rs1805087) are risk factors for NTDs.

Scope of the study

Have RBC folate level and MTHFR and MTR polymorphism anything to do with the severity of the disease in terms of hydrocephalus, lower limb paralysis, and bladder or bowel involvement?

Limitation of the study

Since we have incorporated patients from 0 to 14 years, nutritional deficiency in the form of folate deficiency could be a confounding factor in the estimation of RBC folate level. A newborn age group of such NTD patients would have been a better study population.

Acknowledgments

We are thankful to Director and HOD of Biochemistry, IGIMS, Patna, to grant permission to carry out the work in the present institute.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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    Abstract
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  2005 - Journal of Indian Association of Pediatric Surgeons | Published by Wolters Kluwer - Medknow 

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