Introduction
Down's syndrome (DS) is one of the most common and well-known of all chromosomal abnormalities and accounts for ~30% of all moderate-to-severe cases of mental retardation (1). DS affects approximately one in every 600 live births in the United States (2). Postmortem studies have demonstrated that beginning at age 40, individuals with DS have a significantly higher risk of neuropathological alterations that meet the clinical criteria for Alzheimer's disease (AD) compared with the general population (3). The incidence of AD in DS individuals approaches 15% after age 45 and 76% by age 65 (4). However, the biological mechanisms responsible for these heightened risks remain to be elucidated.
Folic acid (FA), vitamin B12 (VB12) and homocysteine (Hcy) are known biomarkers of the one-carbon unit cycle. FA and VB12 are important in the remethylation of Hcy to form methionine (5). Methionine is an essential amino acid used for protein synthesis or further transformation to S-adenosylmethionine (SAM), which functions as a methyl donor in several methylation pathways, including DNA methylation (Fig. 1) (6). DNA methylation is one of the most important epigenetic mechanisms regulating gene transcription and can result in long-term alterations in cellular function (7). CpG dinucleotide methylation, which is catalyzed by DNA methyltransferases (DNMTs), disrupts the binding of transcription factors and recruits proteins, termed methyl-CpG binding domain proteins, which are associated with chromatin compaction and gene silencing (8). Previous studies have indicated that overall changes in DNA methylation levels may be involved in nervous system degeneration in DS patients (9,10).
The aim of the present study was to compare the levels of one-carbon unit cycle-associated indicators (FA, VB12 and Hcy) in DS patient serum and plasma with those of healthy controls (HCs) and to examine the overall level of DNA methylation in the two groups. The impact of the one-carbon unit cycle on overall levels of DNA methylation was also analyzed in DS children, and preliminary evidence for mechanisms that may be associated with neurodegeneration occurring in children with DS was provided.
Subjects and methods
Subjects and blood collection
Between July 21, 2013 and May 10, 2014, 36 standard pediatric DS children treated at The Children's Hospital of Chongqing Medical University (CHCMU; Chongqing, China) were enrolled in the present study. All individuals were diagnosed with DS by karyo-type analysis of peripheral blood. When standard DS was diagnosed, the following variables were collected: Age, medical history, anthropometric measures and clinical characteristics, including convulsion, cyanosis, strength, pallor, dizziness, bleeding tendency, palpitations, insomnia, decreased heat tolerance, profuse sweating, nervousness, distal tremor, weight loss, diarrhea, hyperdefecation, abdominal distention and recurrent respiratory tract infection. Physical examinations, including somatometry, cardiac examination, respiratory system examination, nervous system examination, thyroid gland and eye examination were performed. In addition, karyotype analysis of peripheral blood and routine blood tests were carried out. These individuals had no complicating medical conditions, including congenital hypothyroidism, gastrointestinal malformations, heart defects, blood diseases, acute respiratory diseases or malnutrition. Individuals found to have the above-mentioned diseases were excluded from the study. A total of 40 age- and gender-matched HCs were recruited from Chongqing's Transportation Bureau Kindergarten (Chongqing, China) and Chongqing Nankai primary school (Chongqing, China), co-operating with the CHCMU. The present study was approved by the ethics committee of CHCMU and informed consent was obtained from the parents of all participants.
Blood samples were collected into drying tubes and EDTA-K2 tubes for serum, plasma and DNA analyses, respectively. The plasma was obtained by centrifuging blood samples at 2,000 × g for 10 min within 30 min of collection. Genomic DNA was extracted from peripheral blood mono-nuclear cells using a Wizard® Genomic DNA Purification kit (Promega, Madison, WI, USA; cat. no. A1125) according to the manufacturer's instructions. Serum, plasma and DNA samples were stored at −80°C for <3 months prior to analysis. The samples were thawed at room temperature prior to analysis.
Determination of serum FA and VB<sub>12</sub> concentrations
FA serum concentrations were measured using a folic acid assay kit (Siemens, East Walpole, MA, USA) on an ADVIA Centaur CP analyzer (Siemens, Nuremberg, Germany) by direct chemiluminescence according to the manufacturer's instructions. VB12 serum concentrations were measured using a vitamin B12 assay kit (Siemens) on an ADVIA Centaur CP analyzer also by direct chemiluminescence according to the manufacturer's instructions.
Plasma Hcy concentration determination
Plasma Hcy concentrations were measured using an homocysteine detection kit (Meikang Biotechnology Co., Ltd., Ningbo, China) and a Hitachi 7600 Automatic Analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan) for enzymatic cycling assays. Hcy concentrations >15 µmol/l were considered to indicate hyperhomocysteinemia (11).
DNA methylation determination
Analysis of the absolute quantity of overall DNA methylation was conducted using a MethylFlash Methylated DNA Quantification kit (Colorimetric; Epigentek Inc., Farmingdale, NY, USA; cat.no. P-1034) according to the manufacturer's instructions. Briefly, DNA (100 ng) was bound to strip wells that were specifically treated to have high DNA affinity. The wells were washed with 150 µl of the 1X wash buffer three times and the capture antibody was added. The wells were washed again and then the detection antibody and enhancer solution was added. Color developing solution was added for color development. Finally, the methylated fraction of DNA was quantified colorimetrically by reading the absorbance in a Synergy H1 Multi-Mode microplate reader (BioTek Instruments, Winooski, VT, USA) at 450 nm within 2 min. The quantity of methylated DNA was proportional to the OD intensity. Absolute overall DNA methylation levels were quantified from a standard curve. The slope of the standard curve was determined using linear regression followed by calculation of the percentage of methylated DNA (5-mC) in total DNA using the following formula (12–14):
5
-
Mc
(
ng
)
=
(
sample
OD
-
ME
3
OD
)
/
(
slope
×
2
)
5
-
Mc
(
%
)
=
[
5
-
Mc
amount
(
ng
)
]
/
S
×
100
%
.
Statistical analysis
Statistical analyses were performed using SPSS 17.0 (IBM, Armonk, NY, USA). One sample Kolmogorov-Smirnov test was used to evaluate the normality of variable distribution. Values with normal distributions are expressed as the mean ± standard deviation (SD), while skewed data are expressed as the median [interquartile interval (IQI)]. Comparison among the groups was performed using an independent t-test for values with normal distribution and a Mann-Whiney U test for skewed variables when appropriate. P<0.05 was considered to indicate a statistically significant difference.
Discussion
Down's syndrome is a chromosomal disorder caused by the presence of three copies of chromosome 21 (15). The increase in the dosage of genes located on this chromosome results in an altered profile of metabolites involved in the folate pathway. These genetic abnormalities can result in folate and VB12 malnutrition, which in turn affects relevant biochemical pathways. Such alterations are attributed to the disturbance of the highly integrated network of metabolic pathways in DS subjects. As a result, cellular dysfunction occurs, which may lead to epigenetic modifications and the consequent unique pathogenesis of Down's syndrome (16).
Individuals with trisomy 21 present with abnormalities in the methionine cycle, which can be attributed to the additional copy of the cystathionine β-synthase (CβS) gene located on chromosome 21 (16,17). The CβS gene encodes an enzyme that catalyzes the condensation of Hcy to form cystathionine in the Hcy transsulfuration pathway, and CβS overexpression leads to an increase in the activity of this pathway. Hcy transsulfuration pathway hyperactivity concomitantly causes an accumulation of 5-methyltetrahydrofolate (5-MTHF) and a reduction in the conversion of 5-MTHF to tetrahydrofolate, which is the metabolically active form of folate required for de novo synthesis of nucleotides necessary for RNA and DNA synthesis. Consequently, a functional folate deficiency can be observed even in the presence of normal or elevated serum folate concentrations (16). The data in the present study are in agreement with this theory. In the present study, with increasing age, serum folate levels decreased significantly, as shown in DS patients in the ≥6 year-old group, which had the lowest FA levels. Therefore, the FA serum level is not an accurate reflection of the FA nutritional status in DS patients. In addition, as DS patients age, the degree of folate deficiency becomes more apparent.
Vitamin B12 is a cofactor that is important for the conversion of Hcy to methionine. Our data revealed that DS patients exhibited lower serum vitamin B12 levels compared with HCs, which are consistent with the results from the study by Meguid et al (18). In addition, the present data demonstrated that the serum levels of FA and VB12 decreased with increasing age in DS patients, with the lowest levels observed in the ≥6 year-old group. Together, these results indicate that DS patients not only show abnormalities in the one-carbon unit cycle, but that the consequences of these abnormalities tend to worsen with age.
Hcy has direct neurotoxicity. In regards to the plasma level of Hcy in DS patients there is certain controversy. Meguid et al (18) reported that DS children had low serum Hcy levels, which is in agreement with the findings of Varga et al (19). However, the present study demonstrated that DS patients had higher levels of Hcy than HCs, which is consistent with the results of Licastro et al (20). It is possible that DS patients may have polymorphisms or altered 5,10-methylene-tetrahydrofolate reductase (MTHFR) activity or expression, and that methionine synthase reductase (MTRR) may contribute to abnormal levels of Hcy (21,22). MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the methyl donor in the remethylation of Hcy to methionine driven by methionine synthase (MS). This in turn is maintained in its active form by MTRR. In addition, MS is a vitamin B12-dependent enzyme. Therefore, reduced MTHFR activity results in an increased demand for FA in order to maintain normal Hcy levels for its remethylation into methionine (23). A previous study demonstrated that Hcy concentrations can be affected by other genetic polymorphisms, including the TCN2 776GG and BHMT 742AA genotypes, which are associated with reduced Hcy concentrations (24). In addition to genetic polymorphisms and alterations in gene expression, the level of FA and/or VB12 also affects the Hcy level. Nutritional intervention with high doses of FA and VB12 in patients with homocysteinemia has proven to be a successful clinical approach to restore normal Hcy levels and folate levels (16,25,26). These findings indicate that plasma Hcy is affected by genotype and nutritional status. However, since these studies included subjects with different ethnicities, ages, genetic polymorphisms and nutritional statuses, drawing broad conclusions regarding the effects of Hcy concentration in DS patients may be difficult.
The folate pathway is important in the interactions between nutrition, epigenomics and gene expression. It also has an effect on DNA methylation, which is one of a group of epigenetic modifications to the genome that affect the expression of specific genes without modifying the sequence of the genome itself. Methyl groups are added to CpG dinucleotides, and these modifications in turn recruit chromatin remodeling complexes that can alter the structure of the surrounding chromatin and either increase or decrease the availability of the gene for expression. Although global DNA methylation levels were higher in DS patients in the present study, the differences were not statistically significant, which is in contrast to the results obtained by Pogribna et al (16). These contrasting results may be due to age and Hcy level differences in the subjects included in the two studies. However, our data suggest that patients with VB12 deficiency show DNA hypermethylation, while patients with hyperhomocysteinemia have DNA hypomethylation. VB12 is required for the conversion of homocysteine to methionine and for the formation of SAM. SAM is involved in biological methylation reactions and allows for the generation of S-adenosylhomocysteine (SAH), which subsequently forms Hcy. SAH is a potent inhibitor of the activity of DNMTs through the product inhibition pathway and can cause genome hypomethylation (27). As such, increased Hcy levels may promote SAH accumulation, which actively inhibits SAM-dependent methyltransferases, including DNMTs, which culminates in DNA hypomethylation. Conversely, decreased VB12 reduces SAH levels, which eventually leads to DNA hypermethylation. Previous studies have demonstrated that alterations in DNA methylation are associated with normal aging and AD (28–30). In addition, the promoter for the gene encoding amyloid precursor protein (APP), which is implicated in the development of AD, is specifically hypomethylated in brain tissues from AD patients (31).
Furthermore, APP is located on chromosome 21 (32). Taken together, these results suggest that alterations in DNA methylation of relevant genes may be involved in the neuropathological alterations observed in DS patients, although additional studies are required to determine the specific methylation sites that are associated with brain aging in DS. In addition, evaluation of changes in epigenetic markers may be useful for identifying biomarkers of brain aging in DS patients.
In conclusion, these data demonstrated that DS patients exhibit abnormalities in the one-carbon unit cycle that tend to worsen with increasing age. DS patients with VB12 deficiency demonstrated DNA hypermethylation, while those with hyperhomocysteinemia had DNA hypomethylation. It was demonstrated that the genetic abnormalities occurring in DS superimpose with VB12 malnutrition in these patients, resulting in the alteration of overall DNA methylation levels that can cause biochemical dysfunction, which in turn may lead to nervous system degeneration. However, further studies are required to identify the specific methylation sites that are associated with brain degeneration in DS patients. Evaluation of alterations in epigenetic markers may be useful for identifying biomarkers of brain degeneration that can occur in DS patients.