Association of MTHFR A1298C polymorphism and blood homocysteine levels with proteinuria in patients with type 2 diabetes mellitus

Abstract

Background

A1298C polymorphism of the MTHFR gene and blood homocysteine levels are reported to be associated with the development of proteinuria in patients with type 2 diabetes mellitus; however, data remain limited.

Objectives

To determine the characteristics of A1298C polymorphism and blood homocysteine levels as well as their association with proteinuria in patients with type 2 diabetes mellitus.

Materials and methods

A cross-sectional study with convenient sampling was performed among patients with type 2 diabetes mellitus who visited the Can Tho University of Medicine and Pharmacy Hospital between August 2023 and August 2024. Blood samples were collected for genetic sequencing and homocysteine level testing. Proteinuria was defined as an albumin-to-creatinine ratio ≥30 mg/g.

Results

In total, 192 patients with a mean age of 63.9 ± 13.1 years were enrolled. Males accounted for 34.9% of the study population. Analysis of A1298C polymorphism revealed genotype distributions of AA, AC, and CC genotypes as 51.6%, 41.1%, and 7.3%, respectively. A significant association was observed between A1298C polymorphism and elevated homocysteine levels, with CC genotype exhibiting a higher prevalence of hyperhomocysteinemia (28.6%) than AA (6.1%) and AC (22.8%) genotypes. Patients carrying AC+CC genotypes had a 2.40-fold higher risk of developing proteinuria (95% confidence interval: 1.30–4.41, p < 0.05) than those with AA genotype. Elevated blood homocysteine levels were associated with an 8.98-fold increased risk of proteinuria (95% confidence interval: 2.06–39.11, p < 0.05). Independent factors associated with proteinuria included age (odds ratio = 0.97), elevated homocysteine levels (odds ratio = 8.79), and AC+CC genotype (odds ratio = 2.08).

Conclusion

A1298C polymorphism was characterized by allele frequencies of 72.1% for A and 27.9% for C. In addition to age, the presence of the C allele and elevated blood homocysteine levels were identified as independent risk factors for proteinuria in patients with type 2 diabetes mellitus.

Keywords

Type 2 diabetes mellitus blood homocysteine levels proteinuria

Introduction

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia due to insulin secretion defects, insulin action impairment, or both.¹ Epidemiological studies have estimated that in 2019, 463 million people worldwide had diabetes, accounting for 9.3% of the global adult population. This number is projected to rise to 578 million (10.2%) by 2030 and 700 million (10.9%) by 2045.² Diabetic renal complications are one of the leading causes of mortality and end-stage renal disease requiring renal replacement therapy in a significant number of patients with T2DM.^1,3 According to Benjamin et al., proteinuria and the albumin-to-creatinine ratio (ACR) are independently associated with an increased risk of various adverse cardiovascular events in patients with T2DM. When ACR exceeds 10 mg/g, cardiovascular risk begins to rise and continues to increase proportionally with ACR levels across different stages of the disease.⁴ Endoplasmic reticulum (ER) stress plays a significant role in the pathophysiology of T2DM.^5,6 Homocysteine (Hcy) is a sulfur-containing amino acid formed during the methylation process that converts methionine to cysteine.⁷ Evidence suggests that Hcy impairs insulin sensitivity in adipose tissue by inducing ER stress and may contribute to the development of diabetic renal complications.⁸ The MTHFR gene encodes the enzyme methylenetetrahydrofolate reductase, which converts methylenetetrahydrofolate to methyltetrahydrofolate and is involved in Hcy metabolism. Therefore, defects in MTHFR can alter plasma Hcy levels.⁹ Increased Hcy levels are associated with the onset of vascular complications in patients with T2DM,¹⁰ with renal complications being relatively common.¹¹ Single nucleotide polymorphisms have been reported to play a significant role in the development of metabolic cardiovascular diseases.¹² A1298C polymorphism of MTHFR affects Hcy metabolism and may also be related to the development of renal complications. The dominant model of A1298C polymorphism has been associated with an increased risk of proteinuria in the study by El-Baz et al.¹³ However, current evidence on the combined effect of MTHFR A1298C polymorphism and Hcy levels on renal complications remains limited. In patients with T2DM, renal complications characterized by the presence of proteinuria have been recognized as being associated with adverse outcomes.^5,6 Understanding the impact of risk factors, such as A1298C polymorphism, may serve as a basis for improving future strategies in disease management and treatment. Therefore, we conducted this study to characterize MTHFR A1298C polymorphism, measure blood Hcy levels, and investigate their association with proteinuria in patients with T2DM.

Materials and methods

Study design and population

This cross-sectional descriptive study employed a convenient sampling method and was conducted among patients who visited and received treatment at the Can Tho University of Medicine and Pharmacy Hospital from August 2023 to August 2024. Inclusion and exclusion criteria were established to select eligible patients for the study. All patients aged >18 years who were diagnosed with T2DM according to the American Diabetes Association criteria¹⁴ were invited to participate. The exclusion criteria were as follows: (a) acute illnesses such as infections or acute kidney injury; (b) acute diabetes complications such as diabetic ketoacidosis or hyperosmolarity; (c) ongoing vitamin B6, vitamin B12, or folic acid supplementation; and (d) psychiatric disorders or dementia. At the end of the follow-up period, a total of 192 patients who met the inclusion and exclusion criteria were enrolled in the study.

Data collection

Data on the following anthropometric characteristics were recorded for all patients: age, sex, duration of diabetes, and body mass index (BMI). A BMI value >23 kg/m² (for Asians) was used to categorize patients as overweight.¹⁵ Waist circumference was measured using a tape measure at the level of the anterior superior iliac spine, with an increased waist circumference in Asians defined as >90 cm for men and >80 cm for women.¹⁶ A 2-mL venous blood sample was collected (with at least 12 h fasting after the previous night’s dinner) for Hcy quantification. Based on the principle of chemiluminescent immunoassay, plasma Hcy level was determined using a one-step immunoassay reaction. The intensity of the emitted light from the chemiluminescent reaction is directly proportional to the level of Hcy present in the sample. Lipid profile components were measured using an enzymatic colorimetric method (using the COBAS-C502 analyzer by HITACHI, Japan). Biochemical test results for glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were recorded, with lipid disorders diagnosed as per the National Cholesterol Education Program Adult Treatment Panel III guidelines.¹⁷ All patients were instructed to void into the toilet as usual upon waking on the first day. For all subsequent urinations throughout the day, patients collected their urine in pre-distributed containers. On the second day, the first morning void was also collected in the container. The total urine collection period was maintained strictly at 24 h, starting from the time of the first urine collection and ending exactly 24 h later. The collected urine samples were delivered to the laboratory on the same day for analysis. The ACR was measured using the COBAS 6000 automated biochemical analyzer. Urinary creatinine level was determined based on the Jaffe method, wherein creatinine forms an orange-colored complex with picric acid in an alkaline medium. The change in absorbance was measured at wavelengths of 520/800 nm and was directly proportional to the creatinine level in the sample. Proteinuria was defined as an ACR of ≥30 mg/g, and macroalbuminuria was identified when the ACR was ≥300 mg/g.¹⁸

Genotyping

Blood samples collected upon admission were transferred to the molecular biology laboratory, where real-time polymerase chain reaction (PCR) was performed to determine MTHFR A1298C polymorphism.

For DNA extraction and purification, 2 mL of blood was collected into a test tube containing ethylene diamine tetraacetic acid anticoagulant. The blood sample was stored and processed at the Laboratory of Can Tho University of Medicine and Pharmacy Hospital. The extraction procedure was performed as per the QiAmp DNA Mini Kit protocol (Qiagen). The DNA pellet was washed with 70% ethanol and then dissolved in 50 mL of distilled water. The DNA concentration and purity were determined by measuring the optical density at 260–280 nm, and purity was verified via electrophoresis on a 1.5% agarose gel. The purified product was stored at −20°C and transported to the sequencing unit under temperature conditions of 4°C–8°C.

After DNA extraction and purification, the samples were tested using the real-time PCR method at the Molecular Biology Laboratory of Can Tho University of Medicine and Pharmacy. In this reaction, the target DNA sequence was amplified, based on the 5′-nuclease principle (TaqMan® method). The Real-time PCR TaqMan® kit from Roche Molecular Systems (USA) was used. Genotyping analysis was performed on the Cobas 5800 system (USA). The results of the genotypic analysis were recorded and subsequently analyzed.

Statistical analysis

The data were analyzed using Statistical Package for Social Sciences 20.0 software. Qualitative variables were presented as frequencies (percentages), while quantitative variables were presented as mean values (±standard deviation (SD)) for data with a normal distribution. The normality of the distribution was tested using the Kolmogorov–Smirnov test and was considered to follow a normal distribution if the p value was greater than 0.05. Differences between qualitative variables were assessed using the chi-square test or Fisher’s Exact test. The difference between two means was compared using the t-test. Finally, logistic regression modeling was employed to identify independent factors influencing the presence of proteinuria in patients with T2DM.

Ethical approval

All patients were informed about the study objectives and methods, following which they provided written consent before participation with the option to withdraw at any stage of the research process. This study was approved by the Ethics Committee in Biomedical Research of the Can Tho University of Medicine and Pharmacy under approval number 23. 057.HV/PCT-HĐĐĐ. This study was conducted in accordance with the principles of the Declaration of Helsinki (1975), as revised in 2024.

Results

In total, 192 T2DM patients (Table 1) with a mean age of 63.9 ± 13.1 years were enrolled. Of these, 34.9% were males. Patients with elevated Hcy levels had a higher mean age than those without (67.9 ± 13.0 vs. 63.2 ±13.1 years, p < 0.05). Similarly, patients with elevated Hcy levels had a lower rate of dyslipidemia than those without (53.6% vs. 73.8%, p < 0.05).

Table 1.

General characteristics of the study population.

Characteristics	Overall(n = 192)	Elevated Hcy (n = 28)	Nonelevated Hcy (n = 164)	p
Male (n (%))	67 (34.9)	10 (35.7)	57 (34.8)	0.922
Age (years) (mean ± SD)	63.9 ± 13.1	67.9 ± 13.0	63.2 ± 13.1	0.028
BMI (kg/m²) (mean ± SD)	23.6 ± 3.7	23.9 ± 3.6	23.5 ± 3.7	0.602
Diabetes duration ≥10 years (n (%))	61 (31.8)	12 (42.9)	49 (29.9)	0.173
Hypertension (n (%))	163 (84.9)	25 (89.3)	138 (84.1)	0.582
Dyslipidemia (n (%))	136 (70.8)	15 (53.6)	121 (73.8)	0.030
Smoking (n (%))	44 (22.9)	7 (25.0)	37 (22.6)	0.777
Glucose (mmol/L) (mean ± SD)	9.2 ± 4.3	9.5 ± 5.0	9.1 ± 4.1	0.660
HbA1c (%) (mean ± SD)	9.2 ± 2.8	9.1 ± 2.9	9.3 ± 2.6	0.859
Triglycerides (mmol/L) (mean ± SD)	2.7 ± 2.1	2.5 ± 1.8	2.8 ± 2.0	0.593
Cholesterol TP (mmol/L) (mean ± SD)	4.7 ± 1.7	4.6 ± 1.1	4.7 ± 1.9	0.847
LDL cholesterol (mmol/L) (mean ± SD)	2.7 ± 1.4	2.7 ± 1.0	2.6 ± 1.4	0.918

SD: standard deviation; BMI: body mass index; HbA1c: glycosylated hemoglobin; TP: total protein; LDL: low-density lipoprotein; Hcy: homocysteine.

Regarding MTHFR A1298C (rs1801131) polymorphism (Table 2) in T2DM patients, the proportions of patients with AA, AC, and CC genotypes were 51.6%, 41.1%, and 7.3%, respectively. Similarly, the frequencies of A and C alleles were 72.1% and 27.9%, respectively. There was no significant difference in A1298C polymorphism between male and female patients (p > 0.05).

Table 2.

Characteristics of MTHFR A1298C polymorphism.

Characteristics	Overall(n = 192)n (%)	Males(n = 67)n (%)	Females (n = 125)n (%)	p
Genotype
AA	99 (51.6)	34 (50.7)	65 (52.0)	0.193
AC	79 (41.1)	31 (46.3)	48 (38.4)
CC	14 (7.3)	2 (3.0)	12 (9.6)
Allele type
A	277 (72.1)	99 (73.9)	178 (71.2)	0.457
C	107 (27.9)	35 (26.1)	72 (28.8)	0.457

There was an association between MTHFR A1298C polymorphism and elevated Hcy levels (Table 3). Patients with CC genotype (28.6%) had a higher rate of elevated Hcy than those with AA (6.1%) and AC (22.8%) genotypes. Similarly, patients carrying the C allele (24.3%) had a higher prevalence of elevated Hcy than those with the A allele (10.8%).

Table 3.

Characteristics of MTHFR A1298C polymorphism and associated factors.

Characteristics	Genotype (n = 192)				Allele type (n = 384)
Characteristics	AA(n = 99)	AC(n = 79)	CC(n = 14)	p	A(n = 277)	C(n = 107)	p
Hypertension	86 (86.9)	65 (82.3)	12 (85.7)	0.671	237 (85.6)	89 (83.2)	0.553
Dyslipidemia	75 (75.8)	51 (64.6)	10 (71.4)	0.259	201 (72.6)	71 (66.3)	0.125
Overweight and obesity	52 (52.5)	41 (51.9)	7 (50.0)	1.0	145 (52.3)	55 (51.4)	0.973
Elevated Hcy level	6 (6.1)	18 (22.8)	4 (28.6)	0.001	30 (10.8)	26 (24.3)	0.028

Hcy: homocysteine.

Patients carrying the risk allele C in the genotypes AC and CC had a 2.40-fold higher risk of developing proteinuria (95% confidence interval (CI): 1.30–4.41, p < 0.05) than those with AA genotype. Furthermore, patients with elevated Hcy levels had an 8.98-fold higher risk of developing proteinuria than those without (95% CI: 2.06–39.11, p < 0.05) (Table 4). Patients with T2DM who developed proteinuria had a higher average Hcy level than those who did not develop proteinuria (10.4 ± 5.3 vs. 8.1 ± 3.1 µmol/L).

Table 4.

Association of MTHFR A1298C polymorphism and blood homocysteine levels with proteinuria in patients with type 2 diabetes mellitus.

Characteristics	Proteinuria		OR(95% CI)	p
Characteristics	Yes (n = 123)	No (n = 69)	OR(95% CI)	p
Genotype
AC+CC (n (%))	69 (56.1)	24 (34.8)	2.40 (1.30–4.41)	0.005
AA (n (%))	54 (43.9)	45 (65.2)	2.40 (1.30–4.41)	0.005
Serum Hyc
Elevated (n (%))	26 (21.1)	2 (2.9)	8.98 (2.06–39.11)	<0.001
Nonelevated (n (%))	97 (78.9)	67 (97.1)	8.98 (2.06–39.11)	<0.001
Hcy level (µmol/L) (mean ± SD)	10.4 ± 5.3	8.1 ± 3.1	–	<0.001

OR: odds ratio; CI: confidence interval; Hcy: homocysteine; SD: standard deviation.

In the multivariate logistic regression model (Table 5), only the factors including age (odds ratio (OR) = 0.97, 95% CI: 0.94–0.99, p < 0.05), elevated serum Hcy (OR = 8.79, 95% CI: 1.81–42.74, p < 0.05), and genotypes containing the risk allele C (AC and CC) (OR = 2.08, 95% CI: 1.04–4.16, p < 0.05) were identified to be independent factors associated with the presence of proteinuria in T2DM patients.

Table 5.

Univariate and multivariate logistic regression analyses of factors associated with proteinuria in patients with type 2 diabetes mellitus.

Characteristics	Univariate		Multivariate
Characteristics	OR (95% CI)	p	OR (95% CI)	p
Age	0.96 (0.93–0.98)	<0.001	0.97 (0.94–0.99)	0.028
Female	2.93 (1.57–5.44)	<0.001	1.41 (0.49–4.02)	0.523
Diabetes duration ≥10 years	2.15 (1.09–4.24)	0.027	1.28 (0.59–2.78)	0.531
Hypertension	1.55 (0.70–3.45)	0.281	–	–
Dyslipidemia	1.13 (0.59–2.18)	0.710	–	–
Smoking	3.53 (1.75–7.10)	<0.001	3.25 (0.99–10.69)	0.052
Overweight/Obesity	1.91 (1.05–3.50)	0.034	1.68 (0.84–3.37)	0.144
Elevated Hcy level	6.72 (2.26–21.02)	0.003	8.79 (1.81–42.74)	0.007
Genotype AC+CC	2.40 (1.30–4.41)	0.005	2.08 (1.04–4.16)	0.039

OR: odds ratio; CI: confidence interval; Hcy: homocysteine.

Discussion

Principal findings

Our study provides important information about MTHFR A1298C (rs1801131) polymorphism in T2DM patients, revealing that the proportions of patients with alleles A and C were 72.1% and 27.9%, respectively, with no significant difference in genotype and allele characteristics between males and females. In addition to age, factors such as carrying the genotype containing the risk allele C and elevated serum Hcy levels were identified to be independent factors that increase the risk of proteinuria in T2DM patients.

Strengths and weaknesses of the study

This study involved a well-designed process of sample collection with clear inclusion and exclusion criteria. All participants voluntarily consented to participate, and genetic sequencing was performed in a modern molecular biology laboratory, ensuring reliable results. The genotypes and alleles of MTHFR A1298C polymorphism in T2DM patients were clearly described and reproducible. In the risk analysis and multivariate model, we identified that both elevated Hcy levels and presence of the genotype with the risk allele C (AC + CC) were associated with the development of proteinuria. This provides important evidence for future studies on this issue.

However, this study was conducted in a single region with a limited sample size, which may have led to differences in some general and clinical characteristics. The cross-sectional design did not account for treatment; thus, the results related to proteinuria may have been confounded by various factors. Therefore, a large-scale multicenter study is required that considers treatment and includes long-term follow-up to provide a more comprehensive understanding of the characteristics of Hcy levels and MTHFR A1298C polymorphism as well as their impact on the development of proteinuria.

Possible explanations and comparison with other studies

T2DM is a metabolic disorder characterized by chronic inflammatory responses, with insulin resistance being a key factor in its pathogenesis. ER stress plays a crucial role in the pathophysiology of this abnormality.^5,6 Evidence suggests that Hcy impairs insulin sensitivity in adipose tissue by inducing ER stress, activating c-Jun N-terminal kinase, promoting the production of proinflammatory cytokines, and facilitating macrophage infiltration. These combined effects contribute to insulin resistance and the onset of T2DM.⁸

At the end of the follow-up period, among the 192 T2DM patients, those with elevated Hcy had a higher mean age than those with normal Hcy levels (67.9 ± 13.0 vs. 63.2 ± 13.1 years). According to Xu et al.,¹⁹ Hcy levels are significantly higher in men than in women, and this trend does not alter with age. The lowest Hcy levels are observed in the 30–50 years age group, with a significant increase observed after the age of 50 years in both sexes. According to Ostrakhovitch and Tabibzadeh,²⁰ the relationship between Hcy level and age is bidirectional. The decline in kidney function, nutritional deficiencies, disturbances in the methionine cycle, and a lack of cofactors for Hcy remethylation and transsulfuration with age all contribute to increased Hcy levels. However, oxidative stress, inactivation of the nitric oxide synthase pathway, and mitochondrial dysfunction also weaken Hcy metabolism, promoting tissue aging.

Regarding MTHFR A1298C polymorphism (rs1801131) in T2DM patients, we observed that the proportions of patients with genotypes AA, AC, and CC were 51.6%, 41.1%, and 7.3%, respectively. Similarly, the allele frequencies for A and C were 72.1% and 27.9%, respectively. These results differ significantly from those of Jafarian et al,²¹ who conducted a study on the Iranian population and reported considerably higher frequencies of the C allele compared with those of the A allele (63.0% vs. 37.0%). The frequencies of genotypes AA, AC, and CC were 4.7%, 64.7%, and 30.6%, respectively. Similarly, Mello et al.²² found that the heterozygous and homozygous genotype frequencies were 27.5% and 5.9%, respectively, in diabetic patients compared with 44.4% and 5.6%, respectively, in the control group. In a study in China by Liu et al.,²³ the frequencies of A1298C polymorphism were 61.35% for AA, 34.38% for AC, and 4.27% for CC, with allele frequencies of 78.54% for A and 21.46% for C. The differences in genotype and allele frequencies between our study and the aforementioned studies may be due to discrepancies in the selection of study populations, geographic regions, and ethnic backgrounds. A1298C polymorphism, located at exon 7, has been reported to reduce the activity of MTHFR, leading to an increase in Hcy levels.⁹ Similarly, we observed a correlation between MTHFR A1298C polymorphism and elevated Hcy levels. Specifically, patients carrying the genotype containing the risk allele C had a higher prevalence of elevated Hcy than the other groups. According to Kumar et al.,²⁴ significantly higher Hcy levels were observed in individuals following vegetarian diets or carrying A1298C polymorphism, particularly the CC genotype. The MTHFR enzyme, the protein product of MTHFR, functions to convert methylenetetrahydrofolate to methyltetrahydrofolate, which is the primary methyl donor in methylation reactions, where Hcy is converted to methionine. Therefore, it can be inferred that defects in MTHFR lead to elevated plasma Hcy levels.⁹

Elevated Hcy levels are associated with the onset of vascular complications in T2DM patients,¹⁰ with renal complications being relatively common.¹¹ MTHFR A1298C polymorphism influences Hcy metabolism, which may also be linked to the development of renal complications. In the study by El-Baz et al., the dominant AC+CC genotype of A1298C polymorphism was associated with an increased risk of proteinuria, with an OR of 1.99 (95% CI: 1.1–3.7, p = 0.037).¹³ This finding is consistent with our results, where both elevated serum Hcy (OR = 8.79) and the AC + CC genotypes (OR = 2.08) were independent factors related to the onset of proteinuria in T2DM patients. However, varying findings have been reported. For example, a meta-analysis by Zhang et al.²⁵ reported that the C allele of A1298C or the CC genotype was not associated with the risk of renal complications (C allele: OR = 0.76, 95% CI: 0.43–1.34, p = 0.34; CC genotype: OR = 1.18, 95% CI: 0.63–2.22, p = 0.60). Interestingly, the AA genotype was associated with a reduced risk of renal complications (OR = 0.68, 95% CI: 0.49–0.96, p = 0.03). Mtiraoui et al.²⁶ conducted an analysis of A1298C polymorphism using PCR–restriction fragment length polymorphism and measured Hcy levels using enzyme-linked immunosorbent assay. Their results indicated that only Hcy was an independent factor contributing to the onset of renal complications, while no association was found between A1298C polymorphism and renal complications. Similarly, Moczulski et al. reported that this polymorphism was not associated with proteinuria or diabetic kidney disease in T2DM patients.²⁷ The underlying mechanism linking MTHFR A1298C (rs1801131) polymorphism to the risk of proteinuria in T2DM patients remains unclear. However, Poduri et al.²⁸ reported that individuals with CC and AC genotypes exhibited a significantly higher prevalence of atherosclerosis than those with AA genotype. This finding suggests that the influence of A1298C polymorphism on the development of proteinuria may be mediated through atherosclerotic processes. Thus, further research is needed to definitively determine the actual impact of A1298C polymorphism on the development of renal complications in T2DM patients.

However, our study has certain limitations. The relatively lower sample size of 192 T2DM patients restricted the ability to conduct subgroup analyses that could more clearly delineate the distribution patterns of genotypes and alleles of MTHFR A1298C (rs1801131) polymorphism in relation to proteinuria, comorbidities, and Hcy levels. Additionally, the absence of a control group limited our capacity to compare the genotypic and allelic characteristics of MTHFR A1298C (rs1801131) polymorphism between patients with and without T2DM. We propose that future studies should address these limitations by conducting large-scale multicenter investigations that include a control group, thereby providing a more comprehensive assessment of the characteristics and potential effects of MTHFR A1298C (rs1801131) polymorphism on proteinuria and Hcy levels in T2DM patients.

Conclusion

This study explored MTHFR A1298C polymorphism in T2DM patients and showed that the proportions of individuals carrying the A and C alleles were 72.1% and 27.9%, respectively. In addition to age, factors such as carrying the genotype containing the C allele and elevated serum Hcy levels are independent factors that increase the risk of proteinuria in patients with T2DM.

Footnotes

Acknowledgements

We would like to thank the Can Tho University of Medicine and Pharmacy for creating favorable conditions for this study to be carried out.

Authorship contribution statement

Nga Thi Ngoc Pham: Writing–review & editing, Conceptualization. Hoang Minh Phan, Hang Thi Thu Ho: Writing–review & editing. Thao Thai Thanh Le: Data curation, Writing–review & editing, Writing–original draft, Data curation. Ha Hong Nguyen: Writing–review & editing, Writing–original draft, Formal analysis, Conceptualization.

Data availability statement

Data are available in a public, open access repository for academic and scientific research purposes.

Declaration of conflicting interests

None to declare.

Funding

None.

ORCID iD

Ha Hong Nguyen

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