Genetic Influences on Plasma Homocysteine Levels in African Americans and Yoruba Nigerians

INTRODUCTION

Homocysteine (HCY) is a sulfur-containing amino acid produced in the metabolism of the essential amino acid methionine. It exists at a critical biochemical juncture between methionine metabolism and the biosynthesis of the amino acids cysteine and taurine. HCY (Fig. 1) is normally metabolized via two biochemical pathways: re-methylation, which converts homocysteine back to methionine, and trans-sulfuration, which converts homocysteine to cysteine and taurine. Abnormally high blood levels of HCY signal a breakdown in this biochemical process, resulting in far-reaching biochemical and life consequences such as increased cardiovascular risks and cognitive decline in Alzheimer’s disease (AD). Increased HCY levels have been associated with cerebral atrophy [1–3] and cognitive impairment [4–7]. Meta-analyses have demonstrated a positive association between increased HCY levels and dementia risk and cognitive function in AD patients [8, 9] although the results have been inconsistent [10–15]. Recent metabolomics studies have revealed that methionine and the pathway leading to cysteine and glutathione production may be dysregulated in AD patients [16] suggesting aberrant methylation processes that can contribute to disease pathogenesis (see the study by Fuso and Scarpa [17] for short review). Most studies with homocysteine have been conducted using participants with European ancestry. Population differences in the role of HCY regulation remains under-investigated. Two studies have employed participants of South Korean [14] and African ancestry [11] and showed positive associations between elevated HCY and increased dementia risk.

Potential mechanisms of HCY effects contributing to cardiovascular diseases and dementias include upregulation of arterial smooth muscle cell collagen production [18], extracellular matrix remodeling [19], potassium channel inhibition [20], microvascular remodeling and increased permeability of the blood-brain barrier by reducing gamma-aminobutyric acid (GABA)-A receptor [21, 22], cytoskeletal remodeling [23, 24], increased cell adhesion [25], and induction of MMP-9 (matrix metalloproteinase-9) activation that can lead to blood-brain barrier dysfunction [26]. HCY can also increase neurotoxicity through overstimulation of N-methyl-D-aspartate receptors [27] and promote apoptosis by increasing DNA damage in neurons [28].

Considering the high estimated heritability (57% ) of HCY [29], it is likely that there is substantial genetic predisposition regulating blood levels of HCY. There are several genome-wide association studies (GWAS) of HCY in clinical samples [30, 31], normal older controls [32] and healthy women [33]. A case-control GWAS in a dementia cohort [34] found the gene MTHFD1L (methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like) that was involved in the folate-pathway. Most genetic studies with HCY levels including these GWAS except one GWAS with Filipino women [33] have investigated genetic factors in participants of European ancestry. Even though HCY increased dementia risk in African ancestry population [11], no studies to date were carried out to identify genetic factors influencing HCY in populations of African ancestry. Here we performed a genetic association analysis to investigate genetic influences on HCY by analyzing data from African American and Yoruba Ibadan Nigerian participants who participated in the Indianapolis-Ibadan Dementia Project.

MATERIALS AND METHODS

RESULTS

DISCUSSION

In order to assess the influence of enzyme-coding genes involved in one-carbon metabolism pathway where HCY is produced, targeted gene association analysis was employed. Main effects of rs28635199 in CBS on HCY may indicate that this gene is a potential genetic risk factor for AD and/or dementia considering that HCY is a potential risk factor for AD and/or dementia. CBS is located at chromosome 21q22.3 and encodes an enzyme catalyzing the conversion of homocysteine to cystathionine as the first step of trans-sulfuration pathway that subsequently produces glutathione, taurine, and alpha-ketobutyrate (αKB). Careful regulation of CBS activity is very important to prevent subsequent disordered conditions. Hyperhomocysteinemia and homocystinuria, characterized by an abnormally upregulated homocysteine, can be caused by CBS deficiency [53, 54]. Upregulation of CBS can cause increased ammonia level, decreased glutathione synthesis and high loss of methyl groups due to increased process of homocysteine through trans-sulfuration pathway instead of re-methylation cycle although clinical outcomes of CBS upregulation can vary individually. In addition, one large-scale GWAS of human blood metabolites [55] identified the minor allele (T) of another SNP (rs2851391) in CBS gene significantly associated with decreased plasma betaine levels, which can reduce AD-like pathological changes and memory impairment induced by HCY [56] while high HCY is known to deplete betaine [57].

Considering that HCY has been shown to be elevated in patients with AD and the minor allele (C) of CSB SNP (rs28635199) was associated with decreased level of HCY in this study, this SNP appears to be a protective variant against elevation of HCY. However, relationship between CBS enzyme and HCY may not be simple and need to be thought from a more complex systems biology perspective [58] considering many other genes and environmental factors that can affect the level of HCY and CBS enzyme. One study found that the CBS 844in68 polymorphism (a 68 bp insertion at 844 in the exon 8) was associated with mild hyperhomocysteinemia and could be a risk factor for AD [59], while another study showed that CBS enzyme level was increased in postmortem brains of Down’s syndrome patients who often develop AD compared to levels in brains of normal individuals and CBS enzyme was located to astrocytes and those surrounding senile plaques in the brains of Down’s syndrome patients [60].

This study is the first GWAS of plasma HCY in samples of African ancestry at risk for AD or other dementia. By analyzing 1,858 plasma homocysteine samples collected from older African Americans and Yoruba Nigerians, we identified one novel genome-wide significant association of SNP (rs6940729) in CD2AP gene. The CD2AP gene is also one of the AD candidate genes for the white European ancestry population and three SNPs (rs9349407, rs9296559, rs10948363) in CD2AP have been identified as susceptibility loci for AD [49–51] and one AD candidate SNP (rs9349407) in this gene was associated with neuritic plaque burden [61]. These SNPs failed replication attempts in other African American cohorts [62, 63]. However, the GWAS SNP in this study is not in strong LD with these SNPs (all pairwise r² <  0.2). The effect of this SNP (rs6940729) as a genetic risk factor for AD and/or dementia needs to be further investigated in larger Africa-originated cohorts.

CD2AP is a protein-coding gene with 18 exons located at chromosome 6p12 and its product is proposed to play important roles in the immune system, endocytosis, cytoskeletal reorganization, cell adhesion, and vesicle trafficking [64–70]. Suppression of CD2AP in an APP transgenic mouse model resulted in decreased Aβ release and lower Aβ₄₂/Aβ₄₀ ratio in brain [71] and a drosophila AD model with knockdown of CD2AP fly ortholog showed enhanced tau toxicity [70]. However, CD2AP mRNA expression was not altered in AD brains [72] although the gene was expressed in brain [71]. These inconsistent findings of CD2AP in relation to gene expression in AD brains and AD relevant biomarkers may imply an indirect influence of CD2AP variation on AD through interaction with other genes/pathways and/or genetic influences on other AD mechanisms including cardiovascular risk factors.

To date, it is unknown how CD2AP affects the level of plasma homocysteine as no study has investigated the relationship between CD2AP and plasma homocysteine. Considering that CD2AP is a regulator of cytoskeletal structure and remodeling, while HCY potentially exerts its effects on cells through effects on cytoskeletal structure and production of reactive oxygen species (ROS), genetic variation in CD2AP may influence the relative sensitivity (resistance) of neuronal cells to the cytoskeletal and ROS effects of HCY, thus impacting the balance of cell life and plasticity and cell death [23, 24]. Another potential mechanism linking CD2AP and HCY may be cell adhesion in which both CD2AP and HCY are involved [70]. Due to lack of studies, potential mechanisms to connect CD2AP and HCY deserve investigation.

Suggestive associations in the GWAS results (Fig. 2) included SNPs near DTWD2 that was associated with subcutaneous adipose tissue (SAT) in European ancestry men [73]. Although visceral adipose tissue is more strongly correlated with metabolic risk factors than SAT [74], SAT can increase cardiometabolic risk which may affect AD risk subsequently [75]. Two variants in DYNC1I1 were associated with fat oxidation and total energy expenditure in the Hispanic population [76], implying genetic influence on cardiovascular risk. JRKL-AS1 was associated with formal thought disorder in schizophrenia in European ancestry cohort [77], but no studies to date reported the association with HCY.

Our enriched pathway analysis indicates HCY as a cardiovascular risk by upregulating arterial smooth muscle cell collagen production [18], remodeling extracellular matrix [19], inhibiting potassium channels [20], and antagonizing GABA-A receptor inducing microvascular remodeling [21] and increasing permeability of blood-brain barrier [22], all of which are related to vascular dysfunction. Among enriched pathways, GABA receptor activation appears particularly interesting in light of data on GABA from reactive astrocyte impairing memory in an AD mouse model [78] and its disruption in AD patients (see the study by Lanctot et al. [79] for review). Subunits of GABA-A receptors were dysregulated in AD brain compared to normal brains [52]. In addition, enhanced GABA-A receptor activity was shown to reduce HCY-induced MMP-9 activation by ERK (extracellular signal-regulated kinase) pathway [26] and plasma level of MMP-9 was shown to be increased in AD patients [80].

This study has some limitations, including unbalanced sample size between cognitively normal controls and demented participants and mixed dementia patients. Due to the small number of demented patients (n = 71), it is difficult to exactly assess diagnostic potential of genetic findings in this study and all finding should be replicated in larger well-balanced cohorts in the future study. The dementia group contained some mixed dementia patients although 65 of 71 patients were diagnosed with AD perhaps limiting this influence. The issue of whether HCY is an AD risk factor and the role of identified SNPs on AD pathophysiology warrants further investigation from a systems biology perspective in a carefully controlled environment considering that biological networks are complicatedly interconnected affecting one another to maintain its homeostasis via potential regulatory feedback networks. This study did not consider B vitamin metabolism in relation to HCY metabolism because B vitamin metabolism is beyond the scope of the present manuscript although this topic is interesting and potentially important. We hope to include this in future studies.

In conclusion, we discovered a novel association of CD2AP with plasma homocysteine in participants with African ancestry and found a new variant in the candidate gene CBS associated with HCY. Many clinical trials investigating HCY reduction as a potential modifiable risk factor employed vitamin B6, B12, and folic acid (B9) to reduce HCY although the clinical benefit is still under debate [81–84]. The inconsistent results of clinical trials may partly be due to unexplained genetic factors influencing HCY. Therefore, the findings in this study merit further investigation regarding their potential role as modifiable AD risk factors.