1. Whole Genome Methylation and MTHFR (C677T) polymorphism in Alcohol Dependence
Bhagyalakshmi Shankarappa; Anirrudh Basu; Shwetha Byrappa; Rashmi Chandra; Sandeep Kandregalu;
Biju Viswanath; Sanjeev Jain; Pratima Murthy; Meera Purushottam
Molecular Genetics Laboratory and Center for Addiction Medicine, Department of Psychiatry,
National Institute of Mental Health and Neurosciences, Bangalore, India.
Aim
To study Whole Genome Methylation and MTHFR polymorphism C677T (rs ) in Alcohol dependent (AD) subjects compared to controls in an Indian population.
Clinical Characteristics of Subjects with ADS
Table1
Simple Withdrawal (%)
Complicated Withdrawal (%)
Delirium Tremens (%)
Seizures (%) 40 60
Present
47.65
32.01
Absent
52.34
67.98
Background
Alcohol consumption is known to be associated with decreased uptake of folate , resulting in folate deficiency and increased homocysteine levels. MTHFR (methylenetetrahydrofolate reductase) a candidate gene for alcohol dependence is involved in carbon metabolism. The common C677T transition on MTHFR (which results in the substitution of valine for alanine at amino acid 223) codes for the thermolabile form which has reduced enzyme activity. MTHFR polymorphism may therefore be associated with physical complications of alcohol, including severe withdrawal. We wanted to assess the relationship between whole genome methylation, homocysteine levels and the MTHFR allele in our Alcohol dependent subjects in order to understand the biology of alcohol dependence.
Table2
Age in years
Age at dependence(yrs)
Average alcohol intake per week (units)
Mean±SD
38.33± 8.9
25.89±7.08
94.08±72.42
Range
19-65
10-61
*12-336
* Four AD subjects with 4-6 units and 1 AD subject with 672 units of consumption was seen.
Global Methylation Levels (%)
Homocysteine Levels
Methods
Individuals (AD subjects (N=319); controls (N=133)) were recruited into the study after obtaining informed consent. DNA isolated from the blood sample was used for genotyping by PCR-RFLP for MTHFR C677T. Whole genome methylation was done in a subset of samples ( AD (N=12); controls (N=12)) by Epigentek Kit. Males who met the criteria for AD (ICD 10) seen at the Centre for Addiction Medicine, NIMHANS were recruited after informed consent and assessed using clinical instruments SSAGA-IV(Semi Structured Assessment for Genetics of Alcoholism-IV), SADQ (Severity of Alcohol Dependence score), MINI (Mini International Neuropsychiatric Interview) (version6) and RCCLI (Revised combined clinical & laboratory index for alcoholic liver disease). Homocysteine levels were estimated by Autoanalyzer (Beckman). MTHFR polymorphism was compared between AD subjects and controls as well as between AD subjects of simple and complicated withdrawal using chi-square analysis.
Normal Range of Homocysteine levels = 5-20umol/L
The increased TT genotype frequency was observed in controls compared to AD subjects (Yates Chi-square = 14.61, P=0.0006) (Table 3). Global methylation levels were lower in AD subjects compared to controls. Homocysteine levels are significantly high in AD compared to normal range (5-20umol/L).
Results
Pre sample - AD subjects sampled at the time of admission.
Post sample - AD subjects sampled after treatment for withdrawal symptoms.
Table3
Folate Pathway and Role of MTHFR
Genotype and Allele Frequencies of MTHFR Polymorphism
Allele Frequency
Genotypes Frequency
Polymorphism ID#
Group 
C-Allele
T-Allele
C/C
C/T
T/T
MTHFR rs
Alcohol dependence (N= 319)
0.92
0.07
278 (0.871) 37
 (0.115) 4
(0.012)
Controls
(N= 133)
0.85
0.15
95(0.714)
35(0.263)
3 (0.022)
Chi-square = 14.61; P =
The polymorphism did not show any association with other clinical parameters like type of withdrawal, presence/absence of DT and seizure.
There is a higher prevalence of thermolabile variant MTHFR 677TT in controls compared to AD subjects, which is showing protective effect against alcohol dependence.
Our observation of increased homocysteine levels and decreased global methylation in alcohol dependent subjects compared to controls is suggestive of impaired single carbon metabolism in these individuals.
Summary and Conclusion
Raphael Saffroy; Amine Benyamine et al., “Protective effect against alcohol dependence of the thermolabile variant of MTHFR” Drug and Alcohol Dependence 96 (2008) 30–36.
Reference
The research is funded by a grant from the Indian Council of Medical Research, New Delhi, INDIA.
Acknowledgement