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Homocysteine (Hcy) is a sulfur-containing non-protein forming amino acid, which occurs naturally in the blood plasma. It is biosynthesized as an intermediate in the one- carbon pathway from methionine, via two main cofactors: S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). SAM acts as an important cosubstrate and is used by the DNA methyltransferase enzymes in transferring methyl groups to the DNA. The product of this reaction, SAH, is then synthesized to Hcy in a reversible manner. The concentrations of Hcy are maintained by two routes; namely: the remethylation pathway, where Hcy is converted back to methionine, and the transsulfuration pathway, where Hcy is converted to cystathionine to form cysteine [1]. A basic illustration of the Hcy pathway is given in Fig. 1.

Figure 1. A simplified figure of homocysteine pathway as present in the liver, which includes

the main enzymes and dietary factors described in this review. DHFR: Dihydrofolate reductase; SHMT: Serine hydroxymethyltransferase; MTHFR: 5,10- Methylenetetrahydrofolate reductase; MSR: Methionine synthase reductase; MS: Methionine synthase; BHMT: Betaine-homocysteine methyltransferase; MAT: Methionine adenosyltransferase; DNMT: DNA methyltransferase; SAHH: S-adenyl-l-homocysteine hydrolase; CBS: Cystathionine β-synthase; CSE: Cystathionine γ-lyase. Light gray boxes: B- vitamins; Dark gray boxes: enzymes; Black boxes: Homocysteine and DNA methylation; the dashed line divides the remethylation from the transsulfuration pathway.

Almost 20 years ago, high concentrations of Hcy in a condition called hyperhomocysteinemia (HHcy), were shown to be an independent risk factor for several disorders including cardiovascular diseases [2] and osteoporotic fractures [3]. Since the reaction from SAH to Hcy is reversible, high concentrations of Hcy increase the concentrations of SAH, which acts as a competitive inhibitor of the methyltransferase reaction [4]. Elevated SAH leads to lower SAM:SAH ratio, which could result in less donation of methyl groups to the DNA by SAM. Elevated SAH is shown to be associated with global DNA hypomethylation, but this phenomenon is tissue-specific and the mechanisms are unknown [5]. Since Hcy is produced as a byproduct of the methyltransferase reaction, alterations in DNA methylation levels are studied as one of the underlying mechanisms of HHcy-associated disorders.

The primary causes of HHcy are dietary and/or deficiencies of the key enzymes of the Hcy metabolism pathway [Fig. 1]. These dietary factors and enzymes that play a role in the remethylation and transsulfuration pathway, balance the concentrations of Hcy by converting it to methionine or cystathionine. The essential dietary factors include methionine and B vitamins (folate, vitamin B6 and vitamin B12). Low vitamin B12 and/or folate are associated with high Hcy [6] and associated risks like cardiovascular disease [7], pregnancy complications [8] and neural tube defects [9]. Besides methionine and B-vitamins, dietary choline and betaine are also important co- factors of the one-carbon pathway. Low dietary choline or with its association with dietary betaine is associated with elevated Hcy in both mice and humans [10–12]. Dietary choline undergoes oxidation to produce betaine and helps in the synthesis of SAM, thus being an indirect methyl donor contributing to the SAM:SAH ratio. Due to limited literature on choline deficient diets, we have focused this review only on high methionine (HM) and B-vitamin deficient diets.

Polymorphisms in genes encoding for enzymes like

methylenetetrahydrofolate reductase (MTHFR), cystathionine β-synthase (CBS), and methionine synthase (MS) are important determinants of Hcy concentrations [13]. MTHFR converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5- MTHF). 5-MTHF acts as a cosubstrate in the remethylation pathway by converting Hcy to methionine. The enzyme MS encoded by the MTR gene, is reductively activated by methionine synthase reductase (MSR) enzyme, which is encoded by MTRR. MS catalyzes the remethylation of methionine from Hcy. The enzyme CBS plays a role in the transsulfuration pathway by catalyzing the conversion of Hcy to cystathionine. Depending on the severity of one or more dietary and/or enzyme deficiencies, HHcy may occur at different levels. Hcy concentrations < 15 μmol/L are referred to as mild, between 15 and 30 μmol/L are referred to as moderate, between 30 and 100 μmol/L as intermediate, and >100 μmol/L as severe HHcy [14].

The focus of this review is to study existing literature on HHcy and its role in relation to DNA methylation. We included studies with effects of diet and/or genotype

on plasma or serum Hcy and its consequent role in the alteration of global, gene- specific or genome-wide DNA methylation. Scientific papers based on both animal and human experiments were reviewed in PubMed. A literature search with varied terms of Hcy and DNA methylation was done, in order to filter out relevant articles which were published from 2001 until 2014. In the animal literature, we focused on mice and rat studies. We included all papers which measured plasma or serum Hcy and either or all of the methylation markers like SAM and SAH or global or gene-specific DNA methylation. We divided the animal literature in three subtopics, namely 1) diet- induced HHcy, 2) genetically-induced HHcy, and 3) genetically- and diet-induced HHcy. A hyperhomocysteinemic (HH) diet mainly involves either HM, low folate (LF) or vitamin B12 or their combination. In addition, low concentrations of choline, riboflavin and pyridoxine might be present. From the human literature, studies which measured plasma or serum Hcy and DNA methylation levels were included, and only if the main aim of the study was to test the association between the two. We focused at vascular diseases, cancer, renal disease and brain disorders.

Methylation can be measured at 3 levels: global, genome-wide and gene- specific. We used the term “global” in this review for studies that report DNA methylation as the total 5-methyl cytosine content, using techniques like cytosine extension assay and LC–MS/MS. Some groups measured methylation of LINE-1, B1 and Alu repetitive elements, which are suggested to be surrogate markers of global methylation levels. We used the term “genome-wide” for methylation measures from DNA-methylation arrays. “Gene-specific” methylation measures methylation levels of cytosines located at specific genes or within their promoters.

2. ANIMAL STUDIES