Global DNA methylation and MTHFR gene polymorphism status of ghanaians with early-onset intracerebral hemorrhage: implications for future interventions

Abstract

Background:

Contrary to global trends, the incidence of intracerebral hemorrhage (ICH) in Sub-Saharan Africa is on a relentless rise, with a relatively early age of onset. While studies among Africans have largely focused on traditional risk determinants, global evidence indicates that genetic and epigenetic variations substantially impact ICH risk.

Aim:

This study aimed to determine the association between MTHFR C677T polymorphisms and ICH risk among Ghanaians with ICH onset at age ≤ 50 years and to compare global DNA methylation levels.

Methods

We conducted a case-control study among 164 ICH cases and 164 matched controls. Genotyping was by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and quality was ensured by including positive and negative controls, with a 100% recall rate. Further validation was through in-silico analysis using National Center for Biotechnology Information (NCBI) reference sequences (https://bit.ly/4kB2KBT). Genotype distribution was categorized as CC = homozygous wild-type, CT = heterozygous, and TT = homozygous variant. Global DNA methylation status was measured using the ab117128 Methylated DNA Quantification Kit.

Results:

The frequency of the CC genotype was 100%, while the CT and TT genotypes were absent in both ICH cases and controls. This limited further assessment of associations. Global DNA methylation status was found to be significantly higher in ICH cases (2.62 ng) compared to controls (2.48 ng), p = 0.030.

Conclusions:

In this Ghanaian cohort, no meaningful evaluation of the association between the MTHFR C677T polymorphism and ICH risk was possible due to its monomorphic distribution. However, increased global DNA methylation in ICH cases compared to controls suggests a potential role of epigenetic mechanisms in the pathophysiology of early-onset ICH, with possible prospects for risk mitigation, particularly in this nutritionally vulnerable population. Further genome- and epigenome-wide association studies are warranted in this population.

Introduction

Sub-Saharan Africa (SSA) has one of the highest age-adjusted prevalence of intracerebral hemorrhage (ICH) globally, with disproportionately higher occurrence of disability and mortality. In contrast to global trends, the age of ICH onset in SSA is also relatively younger [1–3]. These peculiar trends have profound economic implications in resource-constrained settings.

The occurrence of intracerebral hemorrhage is mediated by interactions between multiple factors, either acting independently or synergistically [4]. While studies among indigenous African populations have largely focused on conventional risk factors such as hypertension, there remains an unexplained residual risk of ICH. Folate metabolism and its downstream effects on homocysteine levels and vascular inflammation have been implicated in ICH risk, especially among populations without mandatory supplementation. This risk is influenced both genetically through MTHFR gene polymorphisms and lifelong epigenetic modifications and non-genetically through nutritional intake.

Building on a backdrop of previously established low folate status and consequent disruptions in folate metabolism among stroke survivors in our setting, the current study evaluates the status of the MTHFR gene C677T (rs1801133), a functional polymorphism widely implicated in ICH among various ethnic groups around the world through the disruption of folate metabolism [5, 6]. The study also seeks to assess the global DNA methylation status of ICH cases compared to controls. Findings may provide insights into population-specific genetic susceptibility and inform targeted preventive interventions for this high-risk population, considering the burgeoning field of nutritional epigenetics [7].

Methods

Study design and settings

This was a case-control study, including 164 intracerebral hemorrhage cases and 164 stroke-free controls, recruited from the Komfo Anokye Teaching Hospital (KATH) using consecutive non-probability sampling. Cases included patients with confirmed spontaneous ICH, based on the occurrence of an acute onset of focal neurological deficit, confirmed by positive findings on computed tomography or magnetic resonance imaging, who were aged 18 to 50 years [8, 9]. Patients with stroke subtypes other than ICH or the presence of trauma or hemorrhagic transformation of cerebral infarction were excluded. (Fig. 1)

Fig. 1.

Flow chart for recruitment of cases and controls.

Controls included stroke-free community-based individuals. Each recruited case was matched to a control of the same sex, within a 5-year bound for age range (+/−5 years). Cases were matched to control subjects by age and sex in a ratio of 1:1 using the individual matching method. Stroke-free status was assessed using the American Heart Association 8-item validated questionnaire for verifying stroke status (QVSFS) [10]. Brain imaging was not required for stroke-free verification among controls.

Ethical considerations

Ethical approval was obtained from the Komfo Anokye Teaching Hospital Institutional Review Board (IRB Approval Number: KATH IRB/RR/046/22). All participants were provided informed consent, and in cases where participants were unable to communicate, written consent was obtained from their relatives or legal proxies.

Data collection

Patient’s socio-demographic characteristics, including age and sex (male or female), were collected. The presence of vascular risk factors, including hypertension, dyslipidemia, diabetes mellitus, alcohol use, smoking status, and recreational drug use, was noted.

Neuro-radiological parameters included ICH sites classified as either Lobar (Cortical), or non-lobar (basal ganglia, thalamic, cerebellar, and brainstem) [11].

Genetic analysis

For genetic analysis, 2 mls of venous blood in an EDTA vial was collected. Genomic DNA was isolated from venous whole blood samples using the Quick-DNA Miniprep plus Kit (Zymo Research, USA) following the manufacturer’s protocol. The concentration and quality of DNA were checked with the Implen Nanodrop spectrophotometer. Genotyping was by PCR-RFLP.

PCR was performed with primer sequences: Forward 5′-AGGACGGTGCGGTGAGAGTG-3′, Reverse 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′. The reaction mixture (volume 25 μl/reaction) composed of 12.5 μl of 2x OneTaq^® Master Mix with Standard Buffer (New England Biolabs, USA), 1.5 μl of each primer (10 μm), 2 μl of DNA template, and sterile nuclease-free water (to make up for the volume of 25 μl) was subjected to amplification. The PCR conditions were initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 10 min. Five microliters of amplified product (198 bp fragment) was subjected to electrophoresis using a 3% agarose gel containing ethidium bromide to confirm the amplification.

10 units of Hinf1 (New England Biolabs, USA) was added to 10 μL of amplified DNA, along with 10X buffer (New England Biolabs, USA) and sterile nuclease-free water, and digestion was carried out at 37 °C for 15 min and inactivation at 80 °C for 20 min. The C to T substitution at nucleotide 677 in the MTHFR gene introduces a restriction site for Hinf1 at position 173. The resultant mixture was subjected to electrophoresis using 3% agarose gel containing ethidium bromide. Duplicates were run for each sample with resolution of digested products on 1.5 and 2 agarose gels. Digestion of the C allele (normal) gave an undigested fragment (198 bp), the T allele (mutant) gave 2 fragments (173 and 25 bp), and the CT allele gave 3 fragments (198, 173, and 25 bp). (Supplementary material 1)

The genotyping quality was ensured through the inclusion of positive and negative controls with a recall rate of 100%. Further validation was performed through in silico analysis using National Center for Biotechnology Information (NCBI) reference sequences (https://bit.ly/4kB2KBT). (Supplementary material 2) Genotype distribution was categorized as CC (homozygous wild type), CT (heterozygous), and TT (homozygous variant) based on allelic composition.

Measurement of global DNA methylation status

Global DNA methylation status (5mC content) was measured using the ab117128 Methylated DNA Quantification Kit (Colorimetric). In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. The negative control was an unmethylated polynucleotide containing 50% of cytosine. The positive control was a methylated polynucleotide containing 50% of 5-methylcytosine. The methylated fraction of DNA was detected using capture and detection antibodies and then quantified calorimetrically by reading the absorbance in a microplate spectrophotometer. The amount of methylated DNA is proportional to the OD intensity measured. All samples run in duplicate, and the averages were used for analysis. To quantify the absolute amount of methylated DNA, the standard curve was first generated using the OD values versus the amount of positive control at each concentration point. The equation for the standard curve was then derived using linear regression (Microsoft Excel’s linear regression functions).

Data analysis

The data extracted from case report forms was entered into a pre-designed database using Microsoft Excel 2021. The data was subsequently cleaned and double-checked for errors and then exported to SPSS Version 28 for further validation and analysis.

Categorical variables were summarized as frequencies and percentages. Mean with standard deviation was used for numerical variables with a normal distribution, while median with interquartile range was used for those that were skewed. To compare sociodemographic and clinical characteristics by referral status, the Student’s t-test or Mann-Whitney test and chi-square tests were used for normally or non-normally distributed continuous variables and categorical variables, respectively. The Hardy-Weinberg equilibrium testing was not applicable as the C677T polymorphism was monomorphic in both cases and controls.

Results

This study included 164 patients with CT-confirmed intracerebral hemorrhage and 164 age- and sex-matched stroke-free community-based controls. 67.7% of intracerebral hemorrhage cases as well as controls were male, p = 1.000. The median age of the ICH cases was 44 [IQR 40–48] years, while the median age for the controls was 43 [40–49] years, p = 0.874. 67.1% of cases were hypertensive compared to 8.5% of controls, p < 0.001. Alcohol use was frequent among both cases (29.3%) and controls (22.6%), p = 0.172. Of 164 ICH cases, 24.4% had lobar ICH. (Table 1)

Table 1.

Socio-demographic characteristics.

Variables	ICH cases N = 164 n(%)	Controls N = 164 n(%)	P-value
Age median [IQR], years	44 [40–48]	43 [40–49]	0.874
Gender (Male)	111 (67.7)	111 (67.7)	1.000
Vascular Risk profile
Hypertension	110 (67.1)	14 (8.5)	<0.001
Diabetes	4 (2.4)	4 (2.4)	0.943
Dyslipidemia	21 (12.8)	6 (3.7)	0.756
Alcohol use	48 (29.3)	37 (22.6)	0.172
Recreational drug use	0 (0)	4 (2.4)	0.831
Smoking status	1 (0.6)	5 (3.0)	0.271
ICH location
Lobar	40 (24.4)	NA
Non-Lobar	124 (75.6)	NA
5mC Concentration (ng)	2.62 (9.52)	2.48 (5.11)	0.030

The allele frequencies of C677T in ICH cases and stroke-free control

As summarized in Table 2, the frequency of the CC allele of the C677T MTHFR (rs1801133) SNP in this Ghanaian population was 100% in both ICH cases and controls, while the CT and TT alleles were completely absent. Hardy-Weinberg equilibrium testing was not applicable due to the monomorphic distribution of the C677T polymorphism among both cases and controls. No meaningful evaluation of the association between the MTHFR C677T polymorphism and ICH risk was possible due to its monomorphic distribution.

Table 2.

Allele frequencies of C677T in ICH cases and stroke-free control.

MTHFR C677T alleles (rs1801133)	ICH Cases, N = 164 n(%)	Control, N = 164 n(%)	OR	p-value
CC	164 (100)	164 (100)	NA	NA
CT	0	0	NA	NA
TT	0	0	NA	NA
HWE	χ2 =NA; P= NA	χ2 =NA; P= NA

CC = homozygous wild type.

CT = heterozygous.

TT = homozygous variant.

Global DNA methylation status of ICH cases and stroke-free controls

The global DNA methylation status was found to be higher in intracerebral hemorrhage cases (median 2.62 ng, IQR 0.96–10.48) compared to the age- and sex-matched stroke-free community-based controls (median 2.48 ng, IQR 0.78–5.89), p = 0.030.

Discussion

We evaluated the global DNA methylation and MTHFR C677T polymorphism status of Ghanaians with early-onset intracerebral hemorrhage, compared to stroke-free community-based controls. This represents an initial step towards characterizing the genetic and epigenetic profile of ICH among Ghanaians.

The C677T polymorphism is a common functional polymorphism of the MTHFR gene that controls folate metabolism, homocysteine remethylation, DNA methylation, etc [12]. The MTHFR 677C → T mutation results in a thermolabile MTHFR enzyme with reduced enzyme activity [13]. In the homozygous TT state, the enzyme’s activity is further reduced, leading to a significant disruption of folate metabolism, homocysteinemia, and downstream vascular inflammation, which results in increased ICH risk through vessel fragility and rupture of microaneurysms. Even though the presence of the TT genotype has been confirmed to be prevalent and associated with ICH risk among Asian and Caucasian populations, we found a complete absence of this allele in our Ghanaian cohort [14].

The monomorphic CC profile in our cohort is biologically plausible, as the T allele frequency is extremely low in West Africa [14]. A previous study among healthy Ghanaians residing in Ghana and the UK revealed an overall TT prevalence of 0·5%, highlighting the rarity of the TT genotype in the Ghanaian population [15]. Our results also align with the findings from a recent study conducted among Zambians, which revealed a complete absence of the TT allele, with no significant association with stroke risk [16]. Another study among Moroccan hemorrhagic stroke patients found no statistically significant difference between patients and controls frequencies in the additive, recessive, and dominant models, despite revealing a TT frequency of 10.84% and 9.73% in controls [17]. In this same study, the A1298C gene polymorphisms of MTHFR showed similar distribution, suggesting no association between MTHFR gene polymorphisms and susceptibility to hemorrhagic strokes in Moroccan patients [17]. From an evolutionary standpoint, the principle of negative reproductive selection may partly explain this observation [18]. In populations with folate deficiency, a high prevalence of the TT allele would be expected to result in a more severe phenotype, which may reduce the reproductive fitness, leading to the extinction of the TT allele. In contrast, in populations with mandatory folate supplementation, the phenotypic impact of the mutation is attenuated, allowing greater persistence of the allele. The findings also suggest that genetic impairment of folate metabolism may not significantly drive ICH risk in the Ghanaian population. Considering that low folate status is well established among ICH survivors in our setting, other mechanisms of disruption of folate metabolism resulting from modifiable factors like dietary intake or epigenomic regulation may play a more prominent role in increasing ICH risk in this nutritionally vulnerable population [6].

We observed significantly higher levels of global DNA methylation among ICH cases compared to controls. Although non-specific, global DNA methylation reflects the overall level of DNA methylation and provides insights into the broader epigenomic landscape of an organism. While causality cannot be inferred, this finding suggests possible underlying epigenetic dysregulation in ICH, which may represent either a predisposing mechanism or an acute systemic response to ICH. Emerging epigenome-wide studies in ICH patients show widespread locus-specific differences with a predominance of hypermethylation at promoter and coding regions compared to healthy controls, suggesting increased repression of many genes [19, 20]. Mechanistically, DNA hypermethylation leads to transcriptional silencing of genes, particularly when it occurs at CpG-rich promoter regions, where it interferes with transcription binding and alters chromatin structure [21]. This may inactivate genes required for endothelial repair, nitric oxide production, and other adverse mechanistic pathways, which may increase ICH risk. Further longitudinal studies are needed to clarify the causal and temporal relationships underlying. Interventions prioritizing epigenomic modulation may represent a promising approach to reduce ICH risk mediated by inflammatory vasculopathy in this population [7, 22].

Strengths and limitations

This was a single-center study, which limits its external validity. By design, our study focused on ICH among Ghanaians aged 50 years or less, which may limit the generalization of results to other African populations and to older adults with ICH. Also, the use of a case-control design precludes causal inferences. Additionally, no meaningful evaluation of the association between the MTHFR C677T polymorphism and ICH risk was possible due to its monomorphic distribution in our study sample. Also, our study did not specifically assess non-genetic confounders of the relationship between folate metabolism and ICH risk, such as baseline folate levels, nutrition, access to care, and folate supplementation.

Future directions

Multicentre genome-wide association studies (GWAS) and epigenomic studies are warranted in this high-risk population to provide further insight into the elevated risk of early-onset intracerebral haemorrhage (ICH). Future RCTs evaluating nutritional and epigenetic interventions in reducing ICH risk in the Ghanaian population are also needed to generate evidence for scalable preventive strategies.

Conclusion

The monomorphic distribution of the MTHFR C677T polymorphism in this Ghanaian cohort precluded further assessment of the association with ICH risk in this population. However, increased global DNA methylation in ICH cases compared to controls suggests a potential role of epigenetic mechanisms in the pathophysiology of early-onset ICH, with possible prospects for risk mitigation, particularly in this nutritionally vulnerable population.

Supplementary Material

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.1016/j.neuros.2026.100058.

Abbreviations

GCS

Glasgow Coma Scale

HICs

High-Income Countries

ICH

Intracerebral Hemorrhage

KATH

Komfo Anokye Teaching Hospital

LMICs

Low- and Middle-Income Countries

MTHFR

Methylenetetrahydrofolate Reductase

NCBI

National Center for Biotechnology Information

NHISS

National Institutes of Health Stroke Scale

PCR

Polymerase Chain Reaction

SIREN

Stroke Investigative Research and Educational Network

SNP

Single Nucleotide Polymorphism

SPSS

Statistical Package for the Social Sciences

SSA

Sub-Saharan Africa

Availability of data and materials

De-identified datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.