DNA Methylation change in genes involved in Alcohol metabolism (ALDH2) and One-carbon metabolism (MTHFR) pathways among people with Alcohol Use Disorder

doi:10.21203/rs.3.rs-8049845/v1

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DNA Methylation change in genes involved in Alcohol metabolism (ALDH2) and One-carbon metabolism (MTHFR) pathways among people with Alcohol Use Disorder

https://doi.org/10.21203/rs.3.rs-8049845/v1

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Background: Alcohol Use Disorder (AUD) is a significant public health issue influenced by genetic and environmental factors. Genetic loci linked to age of initiation, response to alcohol, and withdrawal complications.

Alcohol inhibits methionine synthase (MS), disrupting the conversion of homocysteine to methionine and altering levels of S-adenosyl methionine (SAM) and S-adenosylhomocysteine (SAH), which are regulated by DNA methyltransferases (DNMTs). MTHFR, a key enzyme in one-carbon metabolism, and ALDH2, involved in alcohol metabolism, are associated with AUD.

Single nucleotide variants in MTHFR are associated with alcohol withdrawal seizures. Hyperhomocysteinemia has been noted in alcohol-dependent individuals with seizures and brain atrophy. Emerging evidence suggests epigenetic mechanisms may underlie such outcomes.

Methods: The study included 108 male AUD patients (ICD-10 criteria) from St. John’s Medical College Hospital and NIMHANS. Of these, 19 had seizures (SZ + ve), and 89 did not (SZ − ve). Genotyping for ALDH2 (rs2238151) and MTHFR (rs1801133, rs1801131) was done. Plasma homocysteine levels were estimated. DNA was bisulfite converted, and methylation at MTHFR and ALDH2 loci was assessed by pyrosequencing. Statistical tests were carried out to evaluate the results

Results: Significant hypomethylation at 3 CpG sites in MTHFR was seen in SZ + ve vs. SZ − ve (p < 0.01), even after adjusting for alcohol intake. Decreased methylation at ALDH2 loci was noted but not statistically significant.

Conclusion: Changes in methylation levels of MTHFR may perturb this pathway, and thus affect brain function. Altered methylation in blood DNA at candidate loci might serve as a biomarker for prolonged severe alcohol abuse.

Epigenetics & Genomics

Molecular Genetics

Alcohol Use Disorder

DNA Methylation

Alcohol Withdrawal

Alcohol Use Disorder (AUD), as defined by a persistent inability to control alcohol intake despite known adverse consequences, poses a significant challenge to public health. Many factors contribute to the risk of AUD, reflecting a multifaceted interplay between genetic susceptibility and environmental influences, such as the availability of alcohol (Schuckit, 2009). Studies have identified genetic loci associated with distinct alcohol-related traits, including the age of first drink, individual responses to alcohol, and the repercussions of excessive consumption, such as withdrawal symptoms and related complications (Clarke et al., 2017; Huang et al., 2014; Kranzler et al., 2019).

Aldehyde dehydrogenase 2 (ALDH2) plays a crucial role in the metabolism of alcohol by converting acetaldehyde into acetate, that can be easily metabolized via the Krebs pathway (Jain et al., 2019). Variants of the ALDH2 gene, notably the ALDH2*2 allele, that lowers the activity of this enzyme, and leads to an accumulation of acetaldehyde, increases the risk of alcohol-related health issues, particularly in populations of East Asian descent (Eng et al., 2007). Methylenetetrahydrofolate reductase (MTHFR) is essential in one-carbon metabolism, and specific single nucleotide variants (SNVs) in this gene have been linked to a heightened risk of alcohol withdrawal seizures (Lutz, 2008). These seizures are particularly alarming due to their potential to cause severe neurological complications, including brain atrophy and cognitive impairments (Hughes, 2009).

Previous findings suggest that hyperhomocysteinemia is frequently observed in individuals with alcohol dependence, and is associated with both withdrawal seizures and structural brain alterations(Coppola and Mondola, 2018). Another study found that SNPs and DNA methylation in the 1-CM gene influence folate treatment response in hyperhomocysteinemia (HHcy), with DNA methylation mediating the genetic effect(Li et al., 2020). The metabolism of homocysteine is closely regulated by genes involved in one-carbon metabolism, such as MTHFR, indicating that genetic variations in the gene may have significant implications for neurological health in individuals with AUD.

Moreover, epigenetic modifications, including DNA methylation, may play a vital role in the mechanisms contributing to withdrawal seizures. Although it is established that not everyone with AUD experiences these seizures, the underlying reasons for this variability are still not well understood (Gonçalves et al., 2022). Exploring the epigenetic profiles of individuals with AUD, who do experience seizures, could reveal critical distinctions that influence this condition.

This study aims to assess DNA methylation levels in individuals with AUD who have experienced withdrawal seizures. This may help identify potential biomarkers and therapeutic targets that could improve outcomes for those at risk of complicated withdrawal.

Editorial Policies and Ethical Considerations

The study protocol was approved by the Institutional Ethics Committee of St John’s Medical College Hospital (SJMCH) - (IEC Study Ref.No.149/2017) and the National Institute of Mental Health and Neurosciences, Bangalore, India - (NIMHANS/EC(BEH.SC.DIV.) 15th MEETING 2018. Written informed consent was obtained from individuals who were willing to participate in the study. The study procedures were conducted in accordance with the Declaration of Helsinki.

Patients and Samples

Inpatients attending the Departments of Gastroenterology and Psychiatry, St John’s Medical College Hospital (SJMCH); or Centre for Addiction Medicine, National Institute of Mental Health and Neuro Sciences (NIMHANS) from March 2017 to April 2020, seeking treatment for AUD were screened for inclusion in the study. The diagnosis of AUD was made by a psychiatrist with experience in addiction medicine, using Diagnostic and Statistical Manual of Mental Disorders 5 (DSM-5) criteria. Patients with substance use other than nicotine or any major psychiatric and medical disorders based on clinical history and diagnosis were excluded.

The study included hospitalized AUD patients (AUDCe, N = 108) who had been drinking heavily (on average 128 grams of alcohol/day) for several years (8-35 years), Nineteen individuals had developed seizures during withdrawal (SZ+ve), while the others (N=89) had not (SZ-ve).

Genotyping

The genomic DNA was used for genotyping ALDH2 (rs2238151) and MTHFR (rs1801133, rs1801131) polymorphisms as reported earlier(Castro et al., 2004).

DNA Methylation

DNA Methylation for ALDH2 and MTHFR loci

DNA methylation was estimated by pyrosequencing. DNA (450 ng) was subjected to bisulfite conversion (Zymo gold Bisulfite Kit) and amplified using Pyromark PCR kits (Qiagen, Valencia, CA). The ALDH2 and MTHFR methylation analysis were done on the Q24 System (Qiagen) according to manufacturer's instructions. The sequenced region for DNA methylation was in the first intron, and the CpG sites evaluated were close to the transcription start site (TSS : +260 to +289).

Methylation (%5-mC) at five (ALDH2) and three (MTHFR) CpG sites was estimated using Pyro Q-CpG Software (Qiagen). We examined a cytosine at a non-CpG site within PCR products to confirm successful bisulfite conversion. Methylation values in the individual Cytosine-phosphate-Guanine (CpG) sites and the average methylation value across CpG sites were compared across the AUD groups with and without seizures.

Statistical analysis

We described categorical variables using proportions, and continuous variables using mean and standard deviations or median and interquartile range. We checked for normality of continuous variables by Shapiro-Wilk's test (p > 0.05) and carried out post-hoc power analysis.

We used Mann Whitney U tests to analyse differences in DNA methylation levels in both ALDH2 and MTHFR loci (Individual CpG sites) between groups. Following this, we did multiple logistic regression to study the relationship between diagnosis and ALDH2 and MTHFR methylation levels (mean), while correcting for socio-demographic (age) and clinical (age at onset of drinking, quantity, and duration of alcohol use and smoking status) variables and genotype status.

We established that SNP rs2238151 in ALDH2 and rs1801131 in MTHFR were in Hardy Weinberg equilibrium (http://www.oege.org/software/hwe-mr-calc.shtml). We used chi-square tests to analyze differences in genotype and allele frequencies between groups. We also tested for association between clinical variables such as (age at onset of drinking, quantity, and duration of alcohol use) and genotypes using linear regression. All statistical analyses were performed using R version 4.0.2.

The demographic and clinical details of the cases are shown in Table 1. Both groups (SZ+ve and SZ-ve) had similar mean age (43.0 ± 9) years, and had been drinking since early adulthood (AAO 26.0 ± 3 years).

DNA Methylation for ALDH2 and MTHFR loci

DNA methylation data was available for 95 individuals with AUD (SZ+ve=14; SZ-ve=81). At the MTHFR locus we observed significant hypomethylation at all 3 CpG sites (CpG1, p=<0.01; CpG2, p=<0.01; CpG3, p=0.01) in SZ+ve group compared to SZ-ve group (Figure_1). The association remained significant even after correcting for quantity of alcohol consumption and duration of drinking.

At the ALDH2 locus we observed decreased methylation levels in SZ+ve group compared to SZ-ve group but the difference did not reach statistical significance (Figure_2).

Genotyping of ALDH2 and MTHFR SNPs

We did not see any differences in genotype and allele frequencies of ALDH2 (rs2238151) and MTHFR (rs1801131) between SZ+ve and SZ-ve groups.

Homocysteine Levels

No significant association was observed between homocysteine levels and alcohol-related seizures. The mean homocysteine levels in the AUD SZ+ group were 13.42 ± 7.9, while the AUD SZ- group had a mean of 16.17 ± 12.7.

We did not observe any association between variants analysed and alcohol withdrawal seizure.

This study provides evidence that alcohol use and the presence of alcohol withdrawal seizures are associated with significant changes in DNA methylation, particularly at the MTHFR locus. The hypomethylation observed in the SZ+ve group suggests that alcohol-related disturbances in homocysteine metabolism, mediated through methylation changes at the MTHFR locus, could be a mechanism underlying the neurotoxic effects of alcohol. MTHFR plays a crucial role in the remethylation of homocysteine to methionine, and methylation changes in this gene may contribute to altered gene expression, neurodegeneration, and psychiatric symptoms, as suggested by previous studies (Brustolin et al., 2010; Liu et al., 2018; Yang et al., 2020). In particular, elevated homocysteine levels, which can result from MTHFR dysfunction, have been linked to cognitive decline and neuropsychiatric disorders in chronic alcohol users (Hughes, 2009; Zakhari, 2013). The association between hypomethylation of MTHFR and alcohol withdrawal seizures supports the hypothesis that disrupted methylation at this locus may contribute to the neurotoxic effects of alcohol, as the MTHFR gene is integral to neural function and cognitive health (Gales et al., 2018).

While methylation changes at the ALDH2 locus were less conclusive, the data suggest that genetic and epigenetic factors likely contribute to the heterogeneity of alcohol use disorders (AUD) and withdrawal symptoms. ALDH2 plays a key role in alcohol metabolism by converting acetaldehyde to acetate, and its genetic variants, such as the ALDH2 rs2238151 SNP, have been associated with alcohol dependence and the severity of withdrawal symptoms (Schuckit, 2009; Shankarappa et al., 2021). In this study, although we did not find a statistically significant association between ALDH2 methylation and alcohol withdrawal seizures, the observed trend warrants further investigation in larger sample sets to determine whether methylation changes at this locus may influence AUD susceptibility or severity.

Despite the lack of significant findings with respect to ALDH2 and MTHFR SNPs, further research into the broader genetic and epigenetic landscape is needed to fully understand the molecular mechanisms driving AUD and its neuropsychiatric manifestations. Our study did not find significant associations between genetic variants and alcohol withdrawal seizures, consistent with previous work showing ambiguous association between genomic variations at the MTHFR and ALDH2 loci and AUD outcomes (Zuo et al., 2012).

Additionally, the absence of a link between homocysteine levels and alcohol withdrawal seizures suggests that other molecular pathways, beyond homocysteine metabolism, may be involved in the development of withdrawal symptoms. While homocysteine has been identified as a potential neurotoxic agent in alcohol-related disorders (Brustolin et al., 2010; Liu et al., 2018), our findings suggest that other metabolic and epigenetic factors may be at play. This is consistent with studies indicating that multiple pathways, including oxidative stress and neuroinflammation, may contribute to the pathophysiology of alcohol withdrawal (Gonçalves et al., 2022).

Given the complex interplay of genetic, environmental, and epigenetic factors, larger sample sizes and more detailed analyses of additional CpG sites, SNPs, and biomarkers related to homocysteine metabolism may provide deeper insights into the complex relationship between alcohol use, genetics, DNA methylation, and alcohol withdrawal seizures. Studies focusing on a broader set of biomarkers could help clarify the mechanisms underlying the neurotoxicity associated with AUD and alcohol withdrawal (Buchanan and Sinclair, 2021; Heath, 1995; Liu et al., 2018). Attempts to modify DNA methylation in MTHFR or ALDH2 could identify treatment strategies for individuals with AUD, particularly those at risk of severe withdrawal symptoms (Soundararajan et al., 2021; Zakhari, 2013).

The intricate interactions among alcohol, methionine metabolism, and DNA methylation thus offer useful insights into the biological mechanisms that underpin alcohol-related disorders.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding Resources:

1. DBT/PR17316/MED/31/326/2015, Department of Biotechnology, Ministry of Science and Technology, India

2. 3/1/3/42/M/2014-NCD-I, Indian Council of Medical Research

3. IA/CPHI/20/1/505266, The Wellcome Trust DBT India Alliance

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	AUD+veSZ (N=19)	AUD-veSZ (N=89)	P-value
Age (Mean±SD)	43 ± 10	43 ± 9	NS
Years of Drink (Mean±SD)	18 ± 7	16 ± 7	NS
Average alcohol intake (units/day)	16 ± 7	15 ± 7	NS
Age at onset of Drinking (Mean±SD)	26 ± 3	26 ± 2	NS

Table 1. Sociodemographic table for people with Alcohol Use Disorder (N=108).

The authors declare no competing interests.

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DNA Methylation change in genes involved in Alcohol metabolism (ALDH2) and One-carbon metabolism (MTHFR) pathways among people with Alcohol Use Disorder

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