Methylenetetrahydrofolate reductase gene C677T polymorphism and risk of alcohol dependence

Alcohol dependence is a complex neuropsychiatric disorder. Numerous studies investigated association between MTHFR gene C677T polymorphism and alcohol dependence (AD), but the results of this association remain conflicting. Accordingly, authors conducted a meta-analysis to further investigate such an association. PubMed, Elsevier Science Direct and Springer Link databases were searched for studies on the association between the MTHFR C677T polymorphism and AD. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using the fixed- or random-effects model. Statistical analysis was performed with the software program MetaAnayst and MIX. A total of 11 articles were identified through a search of electronic databases, up to February 28, 2020. The results of the present meta-analysis did not show any association between MTHFR C677T polymorphisms and AD risk (for T vs. C: OR = 1.04, 95% CI = 0.88-1.24; CT vs. CC: OR=1.02, 95%CI= 0.62-1.68; for TT + CT vs. CC: OR = 1.10, 95% CI = 0.94-1.29; for TT vs. CC: OR = 1.01, 95% CI = 0.66-1.51; for TT vs. CT + CC: OR = 0.97, 95% CI = 0.66-1.40). Results of subgroup analysis showed no significant association between MTHFR C677T polymorphism with AD in Asian as well as in Caucasian population. In conclusion, C677T polymorphism is not a risk factor for alcohol dependence.

Exact patho-physiological and molecular mechanism of AD is not known yet. However, molecular genetic studies support that multiple genes determine an individual"s predisposition to AD (Begleiter et al., 1999;Sokolov et al., 2003). Heritability of AD likely plays an important role in its development and is determined to be moderate to high (Goldman et al.,2005;Heath et al.,1997). It was reported frequently that alcohol consumption increased homocysteine (Hcy) concentration i.e hyperhomocysteinaemia (Bleich et al.,2005). However, inconsistent results of the combined effect of both positive and negative association have been reported between alcohol intake and Hcy (Ganji and Kafai 2003).
Hyperhomocystenemia is already reported as risk factor for several diseases or disorders including neural tube defects, Alzheimer disease, schizophrenia, pregnancy complications, cardiovascular diseases, noninsulin dependent diabetes and end-stage renal disease as evidenced from several studies (Seshadri et al.,2002).
Homocysteine is a sulfur containing amino acid, several genetic and environmenta ris factor are reported for higher plasma concentration of homocysteine (Gudnason et al.,1998). Homocysteine (Hcy) is synthesized in methionine and folate cycle by demethylation of methionine. 5,10methylenetetrahydrofolate reductase (MTHFR) enzyme of folate cycle plays an important role in homocysteine metabolism. MTHFR gene is present on chromosome 1p36.3. Numerous single nucleotide polymorphisms (SNP) are known in MTHFR gene like C677T andA1298C etc (Frosst et al . 1995;Goyette et al., 1995). The most clinically important and studies polymorphism is C677T (rs 1801133), in which cytosine (C) is substituted with thymine (T) at 677 nucleotide position and consequently alanine is replaced by Valine in MTHFR enzyme(Ala 222 Val) (Rozen et al., 1997;Chango et al., 2000). The variant MTHFR enzyme is thermolabile with reduced activity (`~70%) and it increased the plasma homocysteine concentrations (Frosst et al.,1995). Globally, frequency of mutant T allele varies greatly (Rady et al., 2002;Wilcken et al., 2003;Rai et al., 2010Rai et al., , 2012.  Overall, eleven studies provided 1676/1594 cases/controls for MTHFR C677T polymorphism. The prevalence of C and T alleles in AD cases was 71.22% and 28.79% respectively. The percentage frequency of TT genotype among cases and controls was 9.43% and 12.98%, respectively whereas prevalence of CT heterozygote among AD cases was 38.72% and 39.21% in controls. The prevalence of CC homozygote among AD cases and controls was 51.85% and 47.80%, respectively. Genotypes were in Hardy-Weinberg equilibrium in all controls. In control group the percentage of C and T allele frequencies was 67.41% and 32.59% respectively (Figure 1).

Meta-analysis:
No significant association was observed between the MTHFR C677T polymorphism and the susceptibility to AD in all the genetic models using random effect model ( shows high level of true heterogeneity.

Subgroup analysis:
Out of 11 studies included in the present meta-analysis, 3 studies were carried out in Asian countries, and 8 studies were carried out on Caucasian (Table 1). The subgroup analysis by ethnicity did not reveal any significant association between MTHFR C677T polymorphism and AD in Asian population (T vs.

Publication bias:
Symmetrical shape of Funnel plots" revealed absence of pubication bias. P values of Egger's test were more than 0.05, also provided statistical evidence for the funnel plots" symmetry (p= 0.62for T vs. C; p= . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted June 22, 2020.  Figure 4).

Discussion:
MTHFR enzyme is crucial for the methylation of homocysteine in to methionine and individulas with C677T polymorphism the plasma concentration of homocysteine is high. In vivo and in vitro studies has demonstrated that homocysteine has neurotoxic effects especially on dopamine neurons of reward pathway (Bleich et al . 2000). In addition, hyperhomocysteinemia is also reported in AD (Bleich et al 2000;Cravo and Camilo, 2000). According to deficit hypothesis of addiction, C677T polymorphismdependent alteration of the reward system possibly leads to alcohol addiction.. Further, homovanillic acid (HVA) is a potential indicator of central dopaminergic neuronal activity (Amin et al., 1992) and experimentally, it was demonstrated that hyperhomocystein lowers the eve of HVA in rat stria region (Lee et al., 2005). On the basis of 11 studies providing data on MTHFR C677T genotype and AD risk in two ethnic populations, including over 3,205 subjects, our meta-analysis provides an evidence that TT and CT genotypes or T allele are not associated with AD risk. Hence the present meta-analysis indicated that C677T is a not a risk factor of AD.
Several limitations that should be acknowledged like (i)calculated crude Odds ratio, (ii) included the less number of available studies(10 studies) and the limited sample size of each included study, (iii) observed higher between study heterogeneity, (iv) considered only one gene polymorphism and (v) not considered other confounding factors like diet, gender etc. In addition to limitations, current meta-analysis has several strength also such as -higher study power and larger sample size in comparison to individual case control studies, and absence of publication bias etc.
In conclusion, pooled analysis of data from 11separate articles indicates that the MTHFR 677TT genotype is not a risk factor for AD. The results of present meta-analysis should be interpreted with . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
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(which was not certified by peer review)
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(which was not certified by peer review)
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(which was not certified by peer review)
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(which was not certified by peer review)
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(which was not certified by peer review)
The copyright holder for this preprint this version posted June 22, 2020. . https://doi.org/10.1101/2020.06.15.20132332 doi: medRxiv preprint  . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 22, 2020. . https://doi.org/10.1101/2020.06.15.20132332 doi: medRxiv preprint  . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 22, 2020. . https://doi.org/10.1101/2020.06.15.20132332 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 22, 2020. . https://doi.org/10.1101/2020