deoxyribonucleic acid ( dna ) methylation is a biochemical process catalyzed by dna methyltransferase enzymes to covalently add a methyl group at the 5 position of cytosine dna nucleotides , creating 5-methylcytosine in cpg dinucleotides concentrated in gene promoters .
the cpg dinucleotides in gene promoters are not generally methylated ; thus methylation at these sites is associated with changes in gene expression .
hypermethylation represses transcription , thereby reducing gene expression , while hypomethylation is associated with transcriptional activation of the affected genes [ 2 , 3 ] .
dna methylation is the most characterized of the epigenetic processes , which also include histone modification , chromatin remodeling , and noncoding rnas .
epigenetics , defined as mechanisms that affect gene transcription and/or expression in the absence of alterations to the dna sequence , provides a plausible link between genetic and environmental determinants of health status .
current studies have shown congruence of epigenetic loci and gene polymorphisms in diseases [ 47 ] . in type 2 diabetes , for example , 19 single nucleotide polymorphisms ( snps ) associated with the disease introduced or removed potential sites for dna methylation .
furthermore , the genetic - epigenetic - environment link has been demonstrated in a report that showed an association between the nuclear - encoded gene , nadh dehydrogenase [ ubiquinone ] 1 beta subcomplex subunit 6 ( ndufb6 ) polymorphism , dna methylation , age , and the expression of ndufb6 in human skeletal muscle .
estimates from the international diabetes federation ( idf ) suggest that the population of people with diabetes is growing relatively faster in africa compared to anywhere else .
emerging evidence suggests that this process is not entirely accounted for by the traditional drivers of the diabetes epidemic . in mixed ancestry south africans , for instance , who are largely overweight or obese and at high risk of diabetes ,
the distribution of traditional risk factors for diabetes is not appreciably different between individuals with diabetes and those without diabetes , while the accelerated deterioration of glucose tolerance status over time is not explained by the known powerful determinants of diabetes occurrence . in this context , the contribution of emerging risk factors for diabetes including epigenetic changes has been postulated   but remains largely uninvestigated .
it , therefore , became our primary aim to investigate nontraditional context specific diabetes risk factors and pathophysiological pathways underlying the excess risk of diabetes in this population .
herein , we report the south african mixed ancestry population 's specific differentially methylated sites and metabolic pathways affected by dna methylation in this population .
participants were members of a cohort study conducted in ward 009 , cape town , south africa .
the city of cape town defines ward 009 as a mixed ancestry township formed in the late 1950s . according to the 2011 population census , its population stands at approximately 29,301 with an average household size of 4.84 individuals .
the population is predominantly of mixed ancestry or coloured ( 76% ) followed by black africans ( 18.5% ) and caucasian and asians who make only 1.5% .
most of the residents in this community have lived there for more than five years , while others have been there for their entire lives .
the socioeconomic condition of the people is average with 37% of households having a monthly income of zar3 , 200 or less .
the recruitment of the ward 009 cohort was initiated in april 2014 , from which 3 individuals with screen detected diabetes , 3 individuals with prediabetes , and 3 individuals with normoglycaemia who were age- , gender- , body mass index- ( bmi- ) , duration of residency - matched were selected for the current study .
this investigation is based on the bellville south ( ward 009 ) cohort from cape town that has been approved by the research ethics committees of the cape peninsula university of technology and stellenbosch university ( resp .
the study was conducted according to the code of ethics of the world medical association ( declaration of helsinki ) .
all participants signed written informed consent after all the procedures had been fully explained in the language of their choice .
participants with no history of doctor - diagnosed diabetes mellitus underwent a 75  g oral glucose tolerance test ( ogtt ) as recommended by who .
further , the following biochemical parameters were analyzed at an iso 15189 accredited pathology practice ( pathcare , reference laboratory , cape town , south africa ) : plasma glucose , serum insulin , serum creatinine , total cholesterol ( tc ) , high density lipoprotein cholesterol ( hdl - c ) , triglycerides ( tg ) , low density lipoprotein cholesterol ( ldl ) , c - reactive protein ( crp ) , -glutamyl transferase ( ggt ) , ast , alt , and glycated haemoglobin ( hba1c ) , certified by national glycohemoglobin standardization program ( ngsp ) .
in addition , an edta blood sample was collected and stored at 20 degrees for dna extraction and analysis .
genomic dna was extracted from peripheral blood using the wizard genomic dna purification kit ( promega , madison , wi , usa ) according to the manufacturer 's instructions .
briefly , white blood cells were lysed ; thereafter , cellular proteins were removed by salt precipitation , and high molecular weight genomic dna left in solution was then concentrated and desalted by isopropanol precipitation .
at least 2  g of dna ( concentrations ranging between 70  ng/l and 130  ng/l ) with a260/a280 and a260/a230 ratios  1.8 was shipped frozen on dry ice , as instructed by arraystar inc .
( rockville , md , usa ) according to down et al .   with minor modifications as follows .
for medip , genomic dna was sonicated to ~200900  bp with a bioruptor sonicator ( diagenode , denville , nj , usa ) . thereafter , 800  ng of sonicated dna was end - repaired , a - tailed , and ligated to single - end adapters following the standard illumina genomic dna protocol .
after agarose size selection to remove unligated adapters , the adaptor - ligated dna was used for immunoprecipitation using a human monoclonal anti-5-methylcytosine antibody ( diagenode ) . for this , dna was heat - denatured at 94c for 10  min , rapidly cooled on ice , and immunoprecipitated with 1  l of primary antibody overnight at 4c with rocking agitation in 400  l of immunoprecipitation buffer ( 0.5% bsa in pbs ) . to recover the immunoprecipitated dna fragments , 100  l of protein g magnetic beads ( life technologies , carlsbad , ca
, usa ) was added and incubated for additional 2 hours at 4c with agitation .
after immunoprecipitation , a total of five immunoprecipitation washes were performed with ice - cold immunoprecipitation buffer .
a nonspecific human igg immunoprecipitation was performed in parallel to methyl dna immunoprecipitation as a negative control .
washed beads were resuspended in te buffer with 0.25% sds and 0.25  mg / ml proteinase k for 2 hours at 65c and then allowed to cool down to room temperature .
medip and supernatant dna were purified using qiagen minelute columns and eluted in 16  l eb ( qiagen , germantown , md , usa ) .
fourteen cycles of pcr were performed on 5  l of the immunoprecipitated dna using the single - end illumina pcr primers .
the resulting reactions were purified with qiagen minelute columns , after which a final size selection ( 3001,000  bp ) was performed by electrophoresis in 2% agarose .
libraries were quality controlled with the agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) .
an aliquot of each library was diluted in eb ( qiagen ) to 5  ng/l and 1  l was used in real - time pcr reactions to confirm the enrichment for methylated region .
the enrichment of dna immunoprecipitation was analyzed by qpcr using specific methylated sites at h19 locus and nonmethylated sites at gapdh .
the library was denatured with 0.1  m naoh to generate single - stranded dna molecules and loaded onto channels of the flow cell at 8  pm concentration , amplified in situ using truseq rapid sr cluster kit ( illumina , san diego , ca , usa ) .
sequencing was carried out by running 100 cycles on illumina hiseq 2000 according to the manufacturer 's instructions .
the agilent 2100 bioanalyzer was used for accurate assessment of the quality and concentration of the sequencing library , while the size and concentration of each sample were determined after sequencing library preparation . after the sequencing platform generated the sequencing images , the stages of image analysis and base calling
were performed using off - line basecaller software ( olb v1.8 ) . after passing solexa chastity quality filter ,
the clean reads were aligned to the human genome ( ucsc hg19 ) using bowtie software ( v2.1.0 ) .
statistically significant medip - enriched regions ( peaks ) detected by macs2 were identified by comparison to a poisson background model , using a q - value threshold of 10 .
the peaks in samples were annotated by the nearest gene ( the nearest tss to the canter of peak region ) using the newest ucsc refseq database .
peaks were divided into 3 classes on the basis of their distances to ucsc refseq genes : promoter peaks : promoters were defined as 2000  bp upstream and downstream from the transcription start site ( tss ) .
peaks whose centers were located in these promoter regions were defined as promoter peaks.gene body peaks : the gene body region was defined as + 2000  bp downstream of the transcription start site ( tss ) to the transcription termination site ( tts).intergenic peaks : intergenic regions were defined as the other genomic regions not included in the above 2 regions .
promoter peaks : promoters were defined as 2000  bp upstream and downstream from the transcription start site ( tss ) .
gene body peaks : the gene body region was defined as + 2000  bp downstream of the transcription start site ( tss ) to the transcription termination site ( tts ) .
intergenic peaks : intergenic regions were defined as the other genomic regions not included in the above 2 regions .
individual bases generated from original image files have quality scores , which reflect the probability whether base calling is correct or not .
the chastity ( c ) of each base in the short reads is determined by the intensity of four colours ( ia , ic , ig , and it here ) , and the formula means 
the ratio of the highest ( ic here ) of the four ( base type ) intensities to the sum of highest two ( ic and ig here ) .
 the chastity ( c ) should be no less than 0.6 in the first 25 bases .
the gene ontology project provides a controlled vocabulary to describe gene and gene product attributes in any organism ( http://www.geneontology.org/ ) .
the ontology covers three domains : biological process , cellular component , and molecular function .
fisher 's exact test was used to find if there was more overlap between the de list and the go annotation list than would be expected by chance .
the lower the p value , the more significant the go term ; a p value  0.05 was considered significant .
pathway analysis is a functional analysis mapping of genes to kegg pathways . the p value ( ease score , fisher 's p value , or hypergeometric p value ) denotes the significance of the pathway correlated to the conditions .
the lower the p value is , the more significant the pathway is ; a p value  0.05 was considered significant .
the general characteristics of the nine female participants are presented in table 1 for each participant and further summarized across subgroups defined by the glucose tolerance status .
all participants had reported no menstrual periods for 6 months or more prior to taking part in this study . as expected from the study design ,
hip circumferences were mostly similar across subgroups , while waist circumference and waist - to - hip ratio decreased with improved glucose tolerance .
blood pressure levels were lowest in normotolerant subjects and highest in those with prediabetes . the lipid profile and indicators of glycaemia improved with improving glucose tolerance status , while fasting insulin levels decreased accordingly ( table 1 ) .
a total of 450,142 statistically significant medip - enriched regions ( peaks ) were identified in all the samples . as expected , the promoter region [ tss  2000  bp ; tss + 2000  bp ] showed the least number of peaks , compared to the gene body and intergenic regions , in all groups .
manorm was then used to calculate the statistical significance of differentially methylated regions ( dmrs ) within gene promoters .
subjects with diabetes showed the highest number of dmrs and these are summarized in figure 1 .
generally , more than 80% of dmrs in subjects with diabetes were hypermethylated when compared to those with prediabetes or normoglycaemia , while no differences were observed between subjects with prediabetes or normoglycaemia ( figure 1 ) .
supplementary tables 1 to 6 in supplementary material available online at http://dx.doi.org/10.1155/2016/3172093 show the dmrs in subjects with diabetes , subjects with prediabetes , and controls . to summarize these data , we grouped the dmrs according to chromosomal location and this is shown in figure 2 . compared to controls and subjects with prediabetes ,
hypermethylated dmrs in subjects with diabetes were more common in chromosomes , 3 , 6 , 11 , 13 , and 17 , while in chromosome one , there were more hypomethylated dmrs ( figure 2 ) .
no hypomethylated dmrs were present in chromosome 13 in subjects with diabetes or prediabetes when compared with each other or when those with prediabetes were compared to controls ( figure 2 ) .
we first performed gene ontology ( go ) classification to retrieve the biological process , cellular process , and molecular function of the dmrs and these are presented in supplementary tables 224 , while figure 3 shows biological processes in the top 10 enrichment scores for dmrs in subjects with diabetes or prediabetes . as shown in figure 3 , these hypermethylated dmrs in subjects with diabetes or prediabetes were widely associated with cell surface receptor signaling and inflammatory pathways .
in addition , glucose transport , wnt signaling , muscle development , pancreas development genes , and insulin signaling pathway were associated with hypermethylation in subjects with diabetes or prediabetes ( supplementary tables 712 ) .
although the i - kappab kinase / nf - kappab cascade was associated with hypomethylated dmrs in subjects with diabetes and hypermethylated dmrs in subjects with prediabetes , the genes associated with these pathways were different in each group .
for example , in subjects with prediabetes , the hypermethylated genes were chuk , trim38 , plk2 , tnfrsf19 , and zmynd11 , while in subjects with diabetes they were bcl3 , il23a , f2rl1 , s100a12 , tnfrsf10b , nek6 , rnf31 , slc35b2 , and irak1bp1 . pathway analysis based on the functional analysis mapping of genes to kegg pathways also showed an association with inflammatory pathways ( table 2 ) .
the linoleic acid metabolism and arachidonic acid metabolism pathways were progressively hypomethylated from prediabetes to diabetes . on the other hand ,
the hypertrophic cardiomyopathy ( hcm ) pathway was associated with hypermethylated dmrs in subjects with diabetes when compared to either controls or subjects with prediabetes .
emerging data supports the role of epigenetic mechanisms in the development of diabetes ; however , to date , genome - wide dna methylation profiling has not involved subjects with prediabetes or diabetes from sub - saharan africa or africa in general . in this preliminary genome - wide dna methylation analysis of individuals with prediabetes or diabetes from south africa ,
we provide dmrs data and their biological pathways that appear to be affected in subjects with diabetes or prediabetes , as well as those that appear to show a trend from the prediabetes state to diabetes .
for instance , the linoleic acid metabolism and arachidonic acid metabolism pathways were associated with hypomethylated dmrs in subjects with prediabetes versus controls and in those with diabetes versus those with prediabetes , suggesting that the hypomethylation of these genes is likely to be an early process that occurs before the onset of overt diabetes .
our data also shows that genes related to the immune system , signal transduction , glucose transport , and pancreas development are hypermethylated in subjects with prediabetes or diabetes .
when they are investigated further , these dmrs may be potential biomarkers of disease occurrence / progression and also could suggest possible targets for the development of new treatments .
whole epigenetic profiling in individuals with type 2 diabetes is relatively in its infancy with the first study reported in 2012 .
the study was conducted using pancreatic islets of 5 individuals with type 2 diabetes and identified 276 dmrs , where 96% of the 254 dmrs located in the promoter region were hypomethylated .
the dmrs were associated with beta - cell function , cell death , and adaptation to metabolic stress .
have recently been reported in a study , where they were associated with pathways in cancer , axon guidance ( sema4a and sema5b ) , mapk signaling ( cacna1h ) focal adhesion ( itgb4 ) , ecm - receptor interaction ( agrn and tgb4 ) , and actin cytoskeleton ( tgb4 ) .
furthermore , the study showed an increased accumulation of the dmrs in chromosomes 1 and 2 , while in chromosome 19 , dmrs were lessened . in the current study , we found a total of 1415 dmrs in the promoter regions of subjects with diabetes when compared to control subjects and 81.7% of these were hypermethylated .
's   report , the dmrs were mostly accumulated in chromosomes 1 and 2 but were least in chromosome 21 in subjects with diabetes compared to controls in our study .
we also observed similar finding between subjects with prediabetes and controls showing higher accumulation in chromosomes 1 and 2 , while chromosomes 13 , 16 , 18 , 20 , 21 , and 22 had less than 10 dmrs .
a longitudinal study that investigated hypomethylated dmrs showed that progression from normoglycaemia to a worse glucose tolerance state was associated with early differential methylation prior to disease manifestation .
the authors analyzed methylation levels of candidate dmrs identified by whole epigenetic profiling in 62 subjects with impaired glucose metabolism and 64 controls who maintained a normal glucose tolerance status during follow - up and demonstrated significantly lower percent of methylation before the appearance of the disease in those that progressed .
similarly , we have also observed common dmrs as well as common pathways in prediabetes and diabetes individuals ' inflammatory genes including the lipid metabolism pathway , which appeared to be progressively modified from prediabetes state to diabetes .
we observed that the arachidonic acid ( aa ) pathway was hypomethylated and appeared to be modified in prediabetic and diabetic states ; however , different isoforms of the genes were involved in each glycaemic state .
for example , in subjects with prediabetes versus controls , cytochrome p450 , family 4 , subfamily f , polypeptide 3 ( cyp4f3 ) , cyp4f8 , phospholipase a2 , group iic ( pla2g2c ) , and pla2g4e were differentially methylated , while in subjects with diabetes versus prediabetes , cyp2e1 and pla2g12a were involved .
lipids are important components of all mammalian cells and have a variety of biological functions , including serving as energy reservoirs and mediators of inflammation known as oxylipins .
oxylipins result from the oxygenation of pufas by three types of enzymes , cyclooxygenases , lipoxygenases , and cytochrome p . the type of pufa oxidized and enzyme involved determine the production of oxylipins .
the arachidonic acid ( aa ) generates most of the inflammatory molecules involved in cell signaling cascades and is a precursor of eicosanoids .
eicosanoids include prostaglandins ( pgas ) , leukotrienes ( lts ) , and thromboxanes ( txas ) and their role is dependent on the type of fatty acid from which they are derived . in this regard
, we also observed differentially methylated signals of cytochrome p450 and phospholipase a2 ( pla2 ) .
inflammatory activation of the pla2 enzyme promotes the release of aa from cell membranes phospholipids , which in turn is metabolized by cyclooxygenases ( cox ) , yielding eicosanoids .
taken together , the epigenetic modifications of oxylipins indirectly represent a chronic inflammatory pathway involvement in diabetes development . because fatty acids are derived from the diet in the form of linoleic acid , followed by desaturation and elongation into specific fatty acids ,
the elucidation of lipid pathway epigenetics may contribute to the formulation of treatment and prevention strategies .
for instance , supplement studies using aa , epa , and/or ala have demonstrated an effect on the production of eicosanoids and inflammatory markers . in a study comprising healthy males ,
1.5  g of aa increased pge2 and leukotriene ltb4 , while supplementation with fish oil containing epa and dha decreased the generation of tnf and il-1 by 70% and 78% , respectively . moreover , epigenetic changes are reversible to an extent that in some neurological diseases and cancers epigenetic drugs have been proposed or are currently being used [ 20 , 21 ] . in cancer , for instance , methylation inhibiting drugs include cytidine analogs such as 5-azacitidine   and zebularine . in view of the above ,
it is clear that epigenetics do offer tremendous opportunities for treatment and management of diseases .
considering the current published literature on the epigenetics of type 2 diabetes globally , it is encouraging to note that a few whole epigenomic studies have been conducted in populations from africa .
these include studies that investigated the effect of environment in moroccans , ethiopians , and egyptians [ 2426 ] , severe bladder damage in ghanaians , and exposure to famine in offspring from gambia .
for example , in hapmap lymphoblastoid cell lines derived from individuals of european or african ancestry , population - specific cytosine modifications in samples derived from yoruba people from ibadan , nigeria , and caucasian residents of european ancestry from utah were observed .
the differences between and within population groups have been linked to population - disease - specific single nucleotide polymorphisms ( snps ) . in diabetes , for instance , 17 of the 40 type 2 diabetes candidate genes identified by genome - wide association studies ( gwas ) were differentially methylated in pancreatic islets of subjects with diabetes .
interestingly , in our study , kegg pathway analysis identified hypomethylated dmrs in subjects with diabetes that were associated with african trypanosomiasis pathway .
african trypanosomiasis is a sleeping sickness caused by trypanosoma species from africa , suggesting a population - specific selection of dna methylation in this population with an african ancestry . in this study
, dna methylation was investigated using whole genome medip sequencing ( medip - seq ) ; though bisulfite sequencing is currently considered the gold standard for detecting dna methylation , it does not distinguish between 5-methylcytosine ( 5mc ) and 5-hydroxymethylcytosine ( 5hmc ) . while medip - seq obviates the need for bisulfite treatment of dna
, a limitation of the technique is the inability to detect individual differentially methylated cpg sites .
a major limitation of this study is the lack of verification of regions using other sequencing - based dna methylation profiling methods .
however , a quantitative comparison of four sequencing - based dna methylation methods including medip - seq demonstrated comparable methylation calls of all four methods but differences in cpg coverage , resolution , quantitative accuracy , efficiency , and cost . other limitations of the study include the one gender and small number of individuals investigated .
the pancreatic -cells are believed to be the ideal tissue for type 2 diabetes epigenetics .
we made use of peripheral white blood cells and it has been shown that dna methylation is different between blood cell types ; thus our findings should be interpreted with caution . while we carefully matched the participants , we take note that the smoking patterns were not similar between the groups . therefore , it is likely that some of the dmrs observed are not necessarily due to diabetes or prediabetes but perhaps nicotine .
kg / m ) ; we can not dismiss the fact that some dmrs are obesity related .
although the population investigated is from africa , it is noteworthy to mention that this is a unique heterogeneous group , which is of mixed genetic origin with contributions from europeans , south asians , indonesians , and a population genetically close to the isixhosa sub - saharan bantu population .
therefore we can not rule out the possible role of genetic ancestral components in the methylation patterns observed .
however , financial constraints and the technical complexity of generating ancestry informative markers , particularly for this south african population group , still remain a challenge . in conclusion ,
considering that gwas studies of diabetes involving populations from africa are not available , we recommend methylation quantitative trail loci ( meqtl ) investigations coupled with ancestry informative markers to account for population stratification .
 
 aims . to conduct a genome - wide dna methylation in individuals with type 2 diabetes , individuals with prediabetes , and control mixed ancestry individuals from south africa 
 . methods . 
 we used peripheral blood to perform genome - wide dna methylation analysis in 3 individuals with screen detected diabetes , 3 individuals with prediabetes , and 3 individuals with normoglycaemia from the bellville south community , cape town , south africa , who were age- , gender- , body mass index- , and duration of residency - matched . 
 methylated dna immunoprecipitation ( medip ) was performed by arraystar inc . 
 ( rockville , md , usa ) . results . 
 hypermethylated dmrs were 1160 ( 81.97% ) and 124 ( 43.20% ) , respectively , in individuals with diabetes and prediabetes when both were compared to subjects with normoglycaemia . 
 our data shows that genes related to the immune system , signal transduction , glucose transport , and pancreas development have altered dna methylation in subjects with prediabetes and diabetes . 
 pathway analysis based on the functional analysis mapping of genes to kegg pathways suggested that the linoleic acid metabolism and arachidonic acid metabolism pathways are hypomethylated in prediabetes and diabetes . 
 conclusions . 
 our study suggests that epigenetic changes are likely to be an early process that occurs before the onset of overt diabetes . 
 detailed analysis of dmrs that shows gradual methylation differences from control versus prediabetes to prediabetes versus diabetes in a larger sample size is required to confirm these findings . 
1. Introduction
2. Subjects
3. Materials and Methods
4. Results
5. Discussion