Journal of Applied Hematology

ORIGINAL ARTICLE
Year : 2023  |  Volume : 14  |  Issue : 2  |  Page : 78--86

Predictive effect of methylene tetrahydrofolate reductase variants on vascular related crisis

Suprava Patel1, Rachita Nanda1, Nighat Hussain2, Eli Mohapatra1, Pradeep Kumar Patra3,  
1 Department of Biochemistry, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India
2 Department of Pathology, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India
3 Department of Biochemistry, Chhattisgarh Institute of Medical Sciences, Bilaspur, Chhattisgarh, India

Correspondence Address:
Dr. Suprava Patel
Department of Biochemistry, All India Institute of Medical Sciences, Raipur, Chhattisgarh
India

Abstract

BACKGROUND: Homocysteinemia is regarded as potential predictor for vaso-occlusive phenomenon often observed in sickle cell hemoglobinopathy. The objective was to determine the relationship of these genotypes with homocysteinemia and the predictive coefficient of these polymorphisms on the vascular-related crisis in the presence of sickle cell gene. MATERIALS AND METHODS: The case-control study comprised 89 children diagnosed with sickle disease with features of vascular crisis, 160 children without crisis and 252 apparently healthy children as the control group. The genotypes were assayed for C677T and A1298C variants and their association and predictor effect for homocysteinemia of different grades were analyzed. Sequential multiple regression model was used to assess the predictive effect. RESULTS: Homocysteine levels were significantly higher in the crisis group (P < 0.001). When compared to the wild genotype the variants depicted significantly raised homocysteine levels (P < 0.001). The prevalence of C677T was 29.9% and that for A1298 was 66.3% in the study population. The odds for crisis was 2.3 times for crisis in TT677 and 1.34 times in CC1298 variants. The genotypes revealed a significant association with different grades of homocysteinemia (P < 0.001). Plasma homocysteine depicted significant negative correlation with weight, height, body mass index and hemoglobin levels. None of the TT variants reported normal homocysteine values. Shift toward the variant form showed an increase of homocysteine levels by 7.3 units and 6.9 units for C677T and A1298C single-nucleotide polymorphisms respectively. CONCLUSION: Co-presence of methylenetetrahydrofolate reductase C677T and A1298C polymorphisms could be important predictor for homocysteinemia and thus contribute toward vascular crisis in sickle cell patients.


How to cite this article:
Patel S, Nanda R, Hussain N, Mohapatra E, Patra PK. Predictive effect of methylene tetrahydrofolate reductase variants on vascular related crisis.J Appl Hematol 2023;14:78-86


How to cite this URL:
Patel S, Nanda R, Hussain N, Mohapatra E, Patra PK. Predictive effect of methylene tetrahydrofolate reductase variants on vascular related crisis. J Appl Hematol [serial online] 2023 [cited 2023 Oct 2 ];14:78-86
Available from: https://www.jahjournal.org/text.asp?2023/14/2/78/382411


Full Text



 Introduction



Raised total plasma homocysteine, also referred to as homocysteinemia is regarded as potential predictor for vaso-occlusive phenomenon often observed in sickle cell hemoglobinnopathy. Homocysteinemia can be due to acquired or genetic causes. Acquired causes could be ascribed to modifiable factors like Vitamin B12 or folic acid deficiency which get reversed with supplementation of the nutritional factors.[1] Genetic causes include mutations in the genes coding for the important enzymes of the homocysteine methionine cycle. Mutation in one of such enzymes, has been reported in recent years is methylenetetrahydrofolate reductase (MTHFR) for its potential role in the vascular phenomenon. The enzyme MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyl tetrahydrofolate which is the potential donor for methyl group required for Vitamin B12 dependant remethylation of homocysteine to methionine. Any mutation in the gene coding for the enzyme would reduce the enzyme activity resulting in mild to severe grade homocysteinemia.[2],[3],[4]

Two such single-nucleotide polymorphisms (SNPs) identified as prospective markers for homocysteinemia and vascular crisis are MTHFR C677T and A1298C that occur due to the transition of cytosine © to thymine (T) at 677 and adenine (A) to cytosine (C) at 1298 position. The former replaces valine for alanine and the later glutamate for alanine resulting in reduced enzyme activity.[5],[6] These SNPs have been studied in many diseases such as coronary artery disease, cerebro-vascular diseases, psychiatric disorders, coagulation disorders, cancers, and many more including sickle cell disease.[1],[7],[8],[9] However, there are very limited studies in sickle cell disorder that included both the SNPs and thus, this study would provide a preliminary insight regarding the inter-relationship of both genotypes with homocysteine levels in the children diagnosed with sickle cell hemoglobinopathy.

The said mutation is not uncommon and its co-existence with sickle cell gene mutation behaves as an additive factor increasing the frequency and severity of the vessel-related pathogenesis leading to occlusive crisis. Early screening for this predictor would be quit valuable for risk assessment in these patients. Considering the above-said facts the study was conducted with a primary aim to find the relationship of these genotypes with homocysteinemia and its role as predictor for the crisis events. The secondary aim was to determine the prevalence and understand the distribution of these genotypes in the study population.

 Materials and Methods



The recruitment and enrollment of the children for the study started only after the project was approved by the Institute Ethical Committee. The parents or legally accepted representatives were allowed to go through the participant information sheet and informed consent was duly signed by them before the children were recruited.

This case-control study was conducted on children of age group 5 years to 18 years attending our institute which is a tertiary care hospital. Those who had confirmed reports for the presence of sickle cell hemoglobin, both heterozygous (HbAS) and homozygous (HbSS) variants were included for the study as cases. 89 children with clinical complaints and features of the vascular crisis were enrolled as Group-I and 160 with other complaints but no features of vascular crisis or asymptomatic and had come for follow-up or those with positive HbS report from the control group were included in Group-II. 252 children of 5 years to 18 years who accompanied their parents or siblings and apparently healthy were recruited under the control group. All the children under the control group were analyzed for the presence of HbS by high-performance liquid chromatography (HPLC). Those with HbA and no HbS were included under control group and those with HbS and asymptomatic were categorized as Group-II. None of the study participants were charged for the investigations performed on them.

Children who had a history of blood transfusion in the last 6 months were excluded from the study. In the control group, history for any illness of <6 weeks duration and those with any sort of chronic disorders were excluded from the study. Detailed demographic and clinical history was entered in the prevalidated and ethically approved structured questionnaire for all the children.

Irrespective of the fasting condition and with all necessary aseptic precautions, 5 ml of venous blood was collected in ethylenediaminetetraacetic acid (EDTA) and aliquoted in three different parts. One part was immediately centrifuged to avoid any false rise in homocysteine value due to leakage from the erythrocytes. The separated plasma was aliquoted and stored at − 80°C until analyzed by chemiluminescence method in Advia Centaur immunoanalyzer. The second part of EDTA blood was stored at 2°C–8°C for extraction of deoxyribonucleic acid (DNA) on weekly basis. The third part of the sample was analyzed for hemoglobin (Hb) in Hematology analyzer, Sysmex, and HPLC in D-10 Hemoglobin testing system from Biorad.

DNA was extracted using Invitrogen™ PureLink™ Genomic DNA Mini kit from ThermoFisher Scientific and the extracted DNA was stored at −80°C until processed for polymerase chain reaction (PCR). The assay IDs C_1202883_20 and C_850486_20 were respectively the MTHFR genotyping assay kits for SNPs, C677T (rs1801133), and A1298 (rs1801131) were carried out using TaqMan SNP Assay kit for PCR from Applied Biosystems, ThermoFisher. The real-time PCR was performed in the CFX96 instrument from Biorad.

The study variables such as age, weight, height, body mass index (BMI), blood Hb levels and plasma homocysteine levels were included for analysis. The cut-off reference level for plasma homocysteine was 13 μmol/L (as per kit insert). Those with values above were categorized as homocysteinemia. Plasma levels >13 μmol/L up to 50 μmol/L were considered mild-to-moderate grade homocysteinemia and that with severe rise of >50 μmol/L was considered under severe grade homocysteinemia.[4] Blood Hb above 11 g/dL for cases and above 12 g/dL for the control group was considered normal.[10],[11]Different grades of anemia and homocysteinemia is elaborated in [Table Supplementary 1].[INLINE:1]

The wild, heterozygous, and mutant genotypic forms for C677T were, respectively, CC677, CT677 and TT677 and that for A1298C were AA1298, AC1298, and CC1298.

Statistical analysis

The data were entered and analyzed in IBM©SPSS version 20. The continuous variables were not normally distributed. The variables were transformed to normal distribution before analysis. Mean with standard deviation (SD) of the continuous study variables were compared using analysis of variance within the groups.

Chi-square test with goodness-of-fit tests was applied to see whether the genotypic distribution followed Hardy-Weinberg Equilibrium (HWE) in both control and case groups. Analysis for possible association of the genotypes and different grades of homocysteinemia within the three study groups was performed by Cochran's and Mantel-Haenszel's Chi-square test for linear association. Likelihood ratio with the degree of freedom was used wherever applicable.

Sequential multiple regression (SMR) analysis was carried out to find out whether the presence of SNPs were good predictor for homocysteinemia and thus the disease severity. The SMR technique was applied using three models, each one entered for sets of variables. In the first model, the general variables such as gender, age, and BMI were entered for regression. In model two, the MTHFR SNPs C677T and A1298C were added to those in model 1 and adjusted for the regression coefficient with the other variables. The anemia status and HbS/HbA status were added in addition to those in the above two models to create a third model. This was done to check whether homocysteinemia is solely due to the sickle cell gene or is also influenced by the presence of SNPs and determine the predictor coefficient for homocysteinemia after adjusted for sickle cell status of the study population.

 Results



The study population for this case-control study included 501 children of age group 5–18 years comprising 89 sickle cell cases with vascular crisis, 160 sickle cell cases without vascular crisis and 252 control group.

The mean comparison of study variables

Mean (SD) comparison of the study variables is deciphered in [Table 1]. The children did not differ significantly within the three groups. The mean weight, height, BMI, blood hemoglobin values were significantly lower, whereas plasma homocysteine was greatly elevated in Group-I when compared to Group-II. Most of the study variables in Group-II were comparable with control group except for Hb and homocysteine levels which were significantly different (P < 0.001).{Table 1}{Table 2}

The genotypic distribution of both the SNPs in the three study groups

As outlined in [Table 2], the genotypes for C677T SNP in cases and control were within HWE with P = 0.95 and 0.25 respectively. The overall prevalence of C677T SNP (CT + TT677) in the study population was 29.9% (n = 150) with a prevalence of 28.1% in cases and 31.8% in control group. The three genotypes CT677, TT677 and CC677 were uniformly distributed between cases and control groups (χ2 = 2.59, P = 0.27). Neither the dominant model (CC vs. CT + TT677) nor the recessive model (CC + CT vs. TT) for this SNP differed significantly (P = 0.18 and 0.12) when analyzed for the cases and controls. All three study groups depicted higher frequency of wild genotype CC677. The odds for cases to have crisis was 2.3 times (95% confidence interval: 1.31–4.09, P = 0.002) if TT677 genotype was possessed over wild CC677 when compared to Group-II cases.

The A1298C genotypes also followed HWE in cases and control (P = 0.88 and 0.99). The prevalence of variant genotypes (AC + CC1298) was 66.3% (n = 332) in the study population. The frequency was 69.1% in cases and 63.5% in the control group. Similar to other SNP, the three genotypic forms were more so homogenously distributed among the groups (P = 0.16). Unlike the former SNP, the recessive model (AA + AC vs. CC1298) depicted somewhat significant difference between the cases and controls (P = 0.04). The heterozygous form was more prevalent that the wild form for this SNP. The frequency percentage was highest for the CC1298 variant in Group-I (39.3%). The probability for the vascular-related crisis was increased by 34% in the presence of the CC1298 variant as against the AA1298 genotype (P < 0.001) when compared to the noncrisis group. This could be due to the higher minor allele frequency (1298C allele) in cases (44.4%) than the control group (38.7%).

Comparison of mean homocysteine levels within the three study groups

The mean (SD) of the plasma homocysteine levels within the three genotypes of both SNPs are depicted in [Figure 1]. The levels were significantly higher in both the variants when compared to the wild form (P < 0.001). The homozygous variants reported significantly greater values than the heterozygous forms.{Figure 1}

Comparison of study variables in the allelic population of the study groups

[Figure 2] decodes the distribution of different grades of homocysteinemia in the study groups. 15.5% of the children of the control group depicted mild-to-moderate homocysteinemia whereas none of the children under control group or Group-II depicted homocysteine levels above 50 μmol/L. Although 74.2% of children of Group-I recorded mild to moderate rise, all the children with severe grade homocysteinemia were enrolled under the crisis group. The mean values with quartile ranges for the quantitative variables in control and cases are illustrated in [Figure 3]a and [Figure 3]b. In both groups, BMI was significantly lower in children with C1298 alleles than wild A1298 alleles (p<0.01). Similarly, both groups depicted higher homocysteine levels in children with variant alleles (p<0.001). Blood hemoglobin values were significantly low in both variant forms when compared to the wild forms in the control group (p<0.001). In contrast, the children enrolled under cases did not show major difference in hemoglobin levels between the wild and variant alleles.{Figure 2}{Figure 3}

The frequency percentages of degree of BMI, anemia and homocysteinemia in allelic study population is reflected in [Figure 4]a, [Figure 4]b and [Figure 4]c. Nearly 62% of the children with variant C1298 (p<0.001) and T677 (p=0.03) alleles showed low BMI in cases. When compared to wild allelic group, approximately 20% of children with C1298 and T677 variant alleles revealed moderate degree anemia in control groups (p<0.001). The frequency of different grades of anemia in cases was comparable within the alleleic groups. The frequency for severe grade homocysteinemia was highest in T677 variants (8%) in cases. None of the children in control group recorded severe form of homocysteinemia but the proportion of moderate grade homocysteinemia was more for variant alleles.{Figure 4}

Association of genotypes with different grades of homocysteinemia

The association of the three genotypes of both SNPs with different grades of homocysteinemia is delineated in [Table 3]. Both the SNPs depicted a significant association with homocysteinemia in the three study groups. None of the TT677 variants reported normal homocysteine levels. 25% of them had severely elevated and 75% had mild to moderate rise in homocysteine in Group-I. 100% of children with TT677 form in Group-II and control group revealed mild to moderate elevation. For the children with the A1298C variant, both CC1298C and AC1298 genotypes were found to contribute equally toward mild-to-moderate rise in plasma homocysteine in cases, whereas in control group >50% children with homocysteinemia were CC1298 variant.{Table 3}

Correlation and regression analysis for predictive effect of the genotypes

Plasma homocysteine depicted significant negative correlation with weight, height, BMI, and hemoglobin levels. The regression coefficient and relationship of the predictors for homocysteinemia is reflected in [Table 4]. The variables gender, age, and BMI accounted for 7.6% (R2 = 0.076) variance for homocysteinemia. As depicted in model two, co-presence of anemia and sickle cell gene depicted nearly four-time increase (29.6%) in effect for the outcome of homocysteinemia when adjusted against other variables. Inclusion of both the SNPs, as in model 3 revealed that 15.8% of the variance of elevated homocysteine levels was attributed to their presence when the rest all variables adjusted. Both the genotypes were found to be significant predictor for homocystenemia even after adjusted for sickle cell status (P < 0.001). Any shift towards the variant form would increase the homocysteine levels by 7.3 units and 6.9 units for C677T and A1298C SNPs, respectively. For a child to be a case of sickle cell disorder would tend to have elevated homocysteine levels by 11 units. The regression analysis between the allelic variants with the different grades of BMI, anemia and homocysteinemia is delineated in [Table Supplementary 2]. The control group depicted a significant relationship between variant allele C1298 with low BMI (p<0.001) with an odds of 7.84 (2.2-27.4). In cases, the chances of low BMI in children with C1298 was more than two times as compared to wild allelic forms. The odds for moderate grade anemia in control group children with C1298 was 11.87 (5.68-24.8) when compared to A1298 and 4.02 (1.87-8.65) for T677 as against C677 allele. Only A1298C showed significant association with anemia grades in children enrolled under cases (p=0.012). The variant T677 allele showed significant relationship with homocysteinemia grade in both groups (p<0.001).{Table 4}

 Discussion



In this study, the mean homocysteine levels were significantly elevated in sickle cell crisis (Group-I) and noncrisis (Group-II) groups when compared to control (P < 0.001) [Table 1]. The findings were in agreement to various other studies that documented the significant rise in sickle cell cases. This has been explained by the mechanism of the vaso-occlusive phenomenon for which homocysteine plays crucial role. This metabolite along with its products has been considered as potent thrombogenic agents.[3],[12],[13] They alter the thrombomodulin expression on erythrocytes, cause protein C activation, enhance platelet aggregation, and stimulate thromboxane release. This justifies the reason that severe homocysteinemia was observed in 100% of cases presenting with crisis [Figure 2] while others did not reveal such higher values.

The study results revealed significant association among the three groups with the genotypes [Table 2]. The wild CC677 form was prevalent in the study groups whereas the variant forms of A1298 depicted higher frequency.[14] . The risk for crisis was 2.3 times for TT677 and 1.3 times in CC1298 variants when compared to wild genotypes. In addition, we also observed that the homocysteine levels were significantly varied within the three genotypes of C677T and A1298C [Figure 1] (P < 0.001). Mild to moderate increase was seen in 15.5% of the control population while it was 4–5 times more in Group-II and I [Figure 2]. Presence of mutant alleles in both cases and control groups, recorded significantly elevated serum homocysteine [Figure 3a and 3b]. Previous studies also depicted that plasma homocysteine was significantly raised in cases.[3],[15],[16],[17] The explanation resides in the fact that the mutated enzymes depict reduced activity in presence of either of the polymorphisms and thus play a significant role in homocysteinemia and vaso-occlusive events. The activity of the mutant TT677 genotype is reduced to 20%–30% of normal and that of CC1298 is 60%–70% of wild form. This explains why the levels were higher in TT677 variants.[5],[17],[18],[19] The higher prevalence of CC1298 than TT677 could be a natural barrier as the presence of later one would be more so fatal for these children. Few studies also depicted insignificant contribution towards homocysteinemia and vascular crisis, especially for A1298C and and suggested that presence of this SNP might not predispose to vascular crisis.[20]

Severe grade increase in homocysteine was evidenced in the variant forms of both SNPs in the children enrolled under Group-I and not in others [Table 3]. This indicated that presence of these genotypes have an additive effect on the vascular mechanism of sickle cell hemoglobinopathy.[19],[21],[22],[23] The control group recorded mild-to-moderate homocysteinemia in 50% of subjects with CC1298 and in 100% of children with TT677 variants [Table 3]. Almost 20-25% of control group had homocysteinemia in presence of variants alleles as against 7-10% of those with wild alleles [Figure 4]c, [Table Supplementary 2]. Lower enzyme activity in the variant forms could be the attributing factors unless other influencing factors like nutritional factors were ruled out.[INLINE:2]

The significant positive correlation with age (P < 0.002) and a negative correlation with BMI (P < 0.001) as depicted in [Table 4], is explained by the facts that plasma homocysteine concentration is also regulated by vitamins like folic acid and B12 levels and the requirement of these vitamins increases with age due to physical growth and development.[24] The mean BMI in both cases and control groups with C1298 allele was significantly low [Figure 3]. More than 30% of the control group and 60% of cases with mutant allele (C1298 and T677) had low BMI [Figure 4a]. If the vitamin levels are not optimum the homocysteine concentration would start rising as age advances. Again nutritional deficiency is quite common in India and more so in sickle cell cases even though they take regular folic acid supplementation.[25],[26],[27] However, other nutritional demands are not met unless supplemented with. The demand for these nutritional factors is substantially increased in children with sickle cell hemoglobin that also affects normal growth and thus the BMI shows a downward trend in them. In agreement to the previous study findings, the said the said fact also explains the significantly low Hb level even in control group with mutant alleles (C1298 and T677, [Figure 3]a][23]. The study lack in not quantifying the vitamin levels and diagnosis of megaloblastic anemia but we did not find a significant correlation with anemia in presence of genotypes as shown in [Table 4] (model 3) and thus suggested that the variant forms are the mainstay reason for homocysteinemia in the study population.{Table 4}

The presence of variant forms depicted significant positive correlation [Table 4] as shown in model 2 and model 3 (P < 0.001). The effect of the genotypes did not reveal much change in model 3 even in the presence of the HbS with a coefficient of 7.3 and 6.8 for C677T and A1298C. Each change of a subject from wild to variant form would increase plasma homocysteine by 7.3 units and 6.8 units, respectively. The individual predictor effect of 0.323 and 0.315 (standardized Beta coefficient) of these SNPs was comparable to that of HbS status of 0.542. Hence, it can be suggested that the presence of these genotypes can be considered important predictor for homocysteinemia. Although sickle cell gene is the main reason for a case to develop homocysteinemia and vascular crisis, the co-existence of these genotypes have revealed an additive effect. The combined effect was responsible for 53% variance (R2 in model 3) for homocysteinemia of which nearly 15.8% was contributed by the genotypes alone and 29.6% by the HbS status.

 Conclusion



In the present study, we observed that the mean homocysteine levels were higher in sickle cell cases and the highest level was seen in the crisis group. The different grades of homocysteinemia were dependant on the variant forms of the SNPs and the plasma homocysteine levels were significantly elevated in them. The adjusted predictive effect for the genotypes alone was nearly 16% after adjusted for other co-variates and additive effect was >50%. Hence, the co-presence of MTHFR C677T and A1298C SNPs could be important predictor for homocysteinemia and that contribute toward vascular crisis in sickle cell patients. Although plasma homocysteine estimation remains the mainstay during diagnostic evaluation of these patients, the knowledge of these MTHFR SNPs might aid in further investigations pertaining to sickle cell disease with frequent vascular events. Primary screening could be an alternative way to identify these high-risk individuals to implement measures to delay or prevent crisis conditions[31].

Funding and support

The study was supported by All India Institute of Medical Sciences, Raipur and Chhattisgarh Council of Science and Technology, Raipur, Chhattisgarh, India

Conflicts of interest

There are no conflicts of interest.

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