a recent national cross - sectional survey showed that the overall prevalence of diabetes was estimated to be 11.6% in the chinese adult population .
diabetes is a progressive disease , due in part to the loss of -cell function , with the reduction in function probably commencing 10 to 12 years prior to diagnosis and aggravated by increasing fasting plasma glucose ( fpg ) levels .
furthermore , east asian patients with type 2 diabetes have a higher risk of developing renal complications than europeans , and , with regard to cardiovascular complications , a predisposition for developing strokes .
these results denote that more studies are needed to explain these interethnic differences , and , most importantly , effective strategies are required to facilitate earlier identification and prevention to combat these growing disease burdens , particularly in china .
methylenetetrahydrofolate reductase ( mthfr ) is the main regulatory enzyme for folate / homocysteine metabolism .
mthfr converts 5,10-methylene - tetrahydrofolate ( thf ) into 5-methyl - thf , the dominant circulating form of folate .
the 5-methyl - thf product donates a methyl group to homocysteine in the generation of s - adenosylmethionine , a major source of methyl groups used for dna methylation .
polymorphism of mthfr 677ct leads to a reduction in enzyme activity , resulting in increased concentrations of plasma homocysteine and lower levels of serum folate , and thereby confers a higher risk for stroke , particularly in those with low folate intake .
a recent meta - analysis of case  control studies found that the homocysteine concentration in individuals with type 2 diabetes was significantly higher than that in control subjects ( 0.94  mol / l , 95% confidence interval [ ci ] 0.401.48 ) , and the odds ratio ( or ) associated with type 2 diabetes for mthfr 677tt relative to 677cc was 1.38 ( 95% ci 1.181.62 ) .
our recent study also showed that participants with mthfr 677tt genotype had a higher prevalence of diabetes than those with cc genotype .
furthermore , plasma homocysteine levels were significantly inversely associated with estimated glomerular filtration rate ( egfr ) , and mthfr 677tt genotype was associated with a higher risk for decreased kidney function independent of homocysteine levels . however ,
no prospective studies investigating the association between mthfr 677ct polymorphism , homocysteine , renal function , and new - onset diabetes ( nod ) have been conducted .
furthermore , the frequency of the mthfr 677 tt genotype , which showed marked ethnic variations , was more common in china than in most of the european countries .
the chinese also had higher homocysteine levels , lower folate concentrations , and did not have a policy of mandatory folic acid fortification in food .
these characteristics make the chinese population particularly suited for testing the possible genotype ( mthfr 677ct polymorphism ) and phenotype ( homocysteine and egfr levels)-disease association using the same analytical framework .
therefore , in the current study , we aimed to evaluate the effect of mthfr 677ct polymorphism , homocysteine , and egfr levels on the risk of nod in a rural chinese cohort , and to identify the possible effect modifiers .
our findings may possibly provide insights into the ethnic differences in diabetic complications seen between east asian patients and europeans .
this report followed the strobe ( strengthening the reporting of observational studies in epidemiology ) statement for cohort studies .
study participants were from an epidemiological study of metabolic syndrome conducted during 2003 to 2005 in rural communities ( dongzhi and wangjiang ) in anqing , anhui province of china .
detailed protocol and the details regarding  study population  and  data collection  have been previously described . briefly , 6301 of the study subjects from dongzhi community who received baseline screening examination were invited for a follow - up visit in 2011 , and 2901 ( 46% ) of them responded .
the nonresponders did not differ from the responders substantially with respect to baseline characteristics ( data not shown ) .
this study was approved by the institutional review boards from the nanfang hospital in guangzhou and the institute of biomedicine in the anhui medical university . written informed consent was obtained from each study participant .
fpg , total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein - cholesterol ( hdl - c ) , and homocysteine were measured on the hitachi 7020 automatic analyzer ( hitachi , tokyo , japan ) .
serum creatinine concentrations were determined using an enzymatic method ( sarcosine oxidase - peroxidase  anti - peroxidase ) .
plasma insulin was measured using an enhanced chemiluminescence method on an elecsys 2010 system ( roche , basel , switzerland ) .
mthfr 677ct genotype was determined by taqman assay designed and manufactured by applied biosystems ( foster city , ca ) .
the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated from the fasting concentrations of insulin and glucose using the following formula : fasting insulin ( u / ml )    fasting glucose ( mmol / l)/22.5 .
we excluded participants with self - reported physician diagnosis of diabetes or fpg 7.0  mmol / l at baseline in the final analysis .
nod was defined as fpg 7.0  mmol / l or self - reported physician diagnosis of diabetes at follow - up year .
current alcohol drinking was defined as drinking alcohol at least once per week in the last year .
the question about insomnia was phrased as follows :  do you frequently suffer from insomnia ?  , and a choice of 3 responses ( frequent : almost every week , medium : 13 times per month , and seldom ) was provided .
baseline characteristics are presented as mean or percentage , except for fasting insulin and homocysteine , which are presented as median ( q1q3 ) because of the skewed distribution .
between - group differences in baseline characteristics were tested using the student t test , the signed rank test , or the  test , accordingly .
the effects of mthfr 677ct polymorphism ( cc , ct , and tt ) , homocysteine ( < 10 , 1016 , and 16 
mol / l ) , and egfr ( 120 , 90120 , < 90  ml / min/1.73  m ) on the risks of nod in males and females were estimated using logistic regression models with adjustment for baseline covariates including age ( year ) , baseline fpg ( 5.6 vs < 5.6  mmol / l ) , body mass index ( bmi , 23 vs < 23  kg / m ) , blood pressure ( < 130/85 , 130/85140/90 , 140/90  mm hg ) , tg ( 1.7 vs
mmol / l ) , hdl - c ( < 1.3 [ females]/1.0 [ males ] vs 1.3/1.0 
mmol / l ) , current cigarette smoking , current alcohol drinking , and insomnia ( frequent , medium , and seldom ) .
r software , version 2.15.1 ( http://www.r-project.org ) was used to perform all statistical analyses .
overall , 2901 subjects were revisited . in this report , study participants with self - reported physician diagnosis of cardiovascular disease ( cvd , n  =  28 ) , diabetes ( n  =  10 ) , hypertension ( n  =  124 ) , or with any missing data ( n  =  186 ) regarding baseline values for age , cigarette smoking status , alcohol drinking status , fasting glucose , homocysteine , mthfr 677ct polymorphism or insomnia , or with any missing data ( n  =  61 ) for fpg or self - reported physician diagnosis of diabetes at follow up , or who had an fpg value of 7.0  mmol / l at baseline(n  =  70 ) were excluded .
the prevalence of mthfr 677 cc , 677 ct , and 677 tt genotypes was 36.8% , 47.4% , and 15.8% , respectively .
this population had no significant deviations in genotype distributions from expected hardy  weinberg equilibrium .
those with mthfr 677 tt genotype had significantly higher homocysteine levels ( median , 12.0 in males and 10.1 
mol / l in females ) than those with ct ( 10.4 and 9.1  mol / l ) or cc ( 10.4 and 8.6 
mol / l ) genotype ( p  <  0.05 for either of these genotypes in males or females ) .
furthermore , there was an inverse association between homocysteine and egfr levels in males ( r  =  0.43 , p  <  0.001 ) and females ( r  =  0.63 , p  <  0.001 ) .
the follow - up time ranged from 5.81 to 7.57 years , with a mean of 7.02 ( sd 0.31 ) years .
there were no significant differences in the follow - up time between subjects with and without nod in males ( 6.97 [ 0.31 ] vs 7.02 [ 0.31 ] , p  =  0.137 ) and females ( 7.05 [ 0.34 ] vs 7.03 [ 0.32 ] , p  =  0.506 )
. the baseline characteristics of the study subjects stratified by nod status and sex are summarized in table 1 .
nod patients had significantly higher tg , bmi , insulin levels , and homa - ir compared with those without in both males and females . however , nod patients had significantly higher mthfr 677 tt genotype rates in females only ( table 1 ) .
baseline characteristics according to nod status by sex the incidence rates of nod in males and females were 8.0% and 7.0% , respectively . compared with subjects with mthfr 677 cc genotype , those with tt genotype had a higher risk of nod in females ( or 2.78 , 95% ci 1.395.56 ) but not in males ( 0.80 , 0.401.61 , p for interaction  =  0.008 ) .
< 10  mol / l ) was not associated with nod in males ( 0.88 , 0.421.85 ) or females ( 1.52 , 0.653.57 ) . however
, mildly decreased egfr ( < 90 vs 90120  ml / min/1.73  m ) was associated with nod mainly in males ( 1.96 , 1.013.78 ; females , 0.74 , 0.321.72 , p for interaction  =  0.134 ) ( table 2 ) .
relationship of mthfr 677ct polymorphism , baseline homocysteine levels , and egfr with the risk of nod in the full models , we observed that higher tg , bmi , fpg levels , and insomnia , in addition to mthfr c677 t polymorphism in females , and higher fpg levels and insomnia in addition to lower egfr levels in males remained independent risk factors for the risk of nod ( data not shown ) . in the stratified analyses ,
the mthfr 677ct polymorphism ( cc , ct , and tt genotypes ) was more strongly associated with the risk of nod among females with higher bmi ( 23 vs < 23  kg / m , p for interaction  =  0.009 ) or lower hdl - c levels ( < 1.3 vs 1.3  mmol / l , p for interaction  =  0.015 ) .
however , the association of mthfr 677ct polymorphism with increased risks of nod in females appeared to be similar among subgroups classified according to baseline homocysteine ( 10 vs < 10  mol / l , p for interaction  =  0.061 ) , egfr ( < 110 vs 110  ml / min/1.73  m , p for interaction  =  0.229 ) , fpg ( 5.6 vs
< 5.6  mmol / l , p for interaction  =  0.635 ) , tg levels ( 1.7 vs < 1.7  mmol / l , p for interaction  =  0.189 ) , or insomnia ( frequent vs medium or seldom , p for interaction  =  0.831 ) . furthermore
, similar trends for egfr category and increased risk of nod in males were observed among subgroups stratified by insomnia ( p for interaction  =  0.946 ) , baseline fpg ( p for interaction  =  0.977 ) , homocysteine levels ( p for interaction  =  0.573 ) , or mthfr 677ct polymorphism ( p for interaction  =  0.880 ) ( table 3 ) .
stratified analysis of multivariate ors for nod among 1172 women according to mthfr 677ct polymorphism further adjustment for homa - ir ( n  =  2144 ) did not substantially change the relationship of mthfr 677ct polymorphism ( tt vs cc , 2.20 ; 1.024.73 ) in females or egfr levels ( < 90 vs 90120  ml / min/1.73  m , 1.85 ; 0.923.71 ) in males with the risk of nod .
previous studies have shown conflicting results regarding the homocysteine levels in patients with diabetes . a recent meta - analysis of 14 case  control studies found that the mean homocysteine concentration was greater in patients with type 2 diabetes than in control subjects . however , the 3 studies that contributed most to the overall estimate included patients with type 2 diabetes accompanied by varying degrees of kidney disorders .
these results suggest that it may be renal dysfunction but not diabetes that mostly explains the difference in homocysteine levels between type 2 diabetes patients and control subjects in this meta - analysis .
in fact , in our present prospective study , we did not find significant associations between homocysteine levels and nod in males or females .
however , consistent with this meta - analysis , female subjects with tt genotype in the current study had a higher risk of nod ( tt vs cc genotype , 2.78 ; 1.395.56 ) . to explain the significant relationship of mthfr gene 677ct polymorphism ( but not homocysteine levels ) with the risk of nod , we speculate that , first , the homocysteine levels ( median 10.7 
mol / l in females ) in the present study was possibly not high enough to cause obvious organ damage .
second , plasma homocysteine may just serve as a reliable functional marker of folate status , and folate may have other actions , including antioxidant actions , effects on cofactor availability , or direct interactions with the enzyme endothelial no synthase , in addition to homocysteine lowering that influence health status .
a previous report showed that folic acid given to patients with coronary heart disease resulted in improvement in endothelial function without any change in plasma homocysteine concentration .
unfortunately , we were not able to directly examine the association between mthfr 677ct polymorphism , folate , and the risk of nod in the current study due to the lack of baseline folate data .
additional studies are required to further address this topic and to evaluate the role of folic acid supplementation in reducing the risk of diabetes , particularly in populations without folic acid fortification .
meanwhile , we did not have enough information to explain the sex - specific effect of mthfr 677ct polymorphism , which may relate to the possible role of hormones in folate / homocysteine metabolism .
our current study detected a detrimentally interactive effect between the mthfr 677ct polymorphism and higher bmi ( 23 vs < 23 
kg / m ) , or lower hdl - c ( < 1.3 vs 1.3 
obese subjects without diabetes have been shown to exhibit an enhanced rate of glucose production .
we hypothesize that  a higher bmi state  may augment the mthfr 677ct polymorphism - mediated risk of nod .
furthermore , mthfr 677ct polymorphism appeared to modify the efficacy of the drug pravastatin in reducing risk of cardiovascular events .
a significantly protective effect against coronary heart disease ( hazard ratio [ hr ] 0.71 , 95% ci 0.580.87 ) was shown in subjects with cc genotype but not in subjects with ct ( hr 1.25 , 95% ci 0.971.61 ) or tt genotype ( hr 0.80 , 95% ci 0.501.28 , p for interaction  =  0.004 ) .
consistently , we also observed an interaction between the mthfr 677ct polymorphism and hdl - c levels on the risk of nod .
these results suggest that an investigation of the possible modifying effect of mthfr 677ct polymorphism on cvd associated with hdl - c increasing therapy maybe provide some clues regarding the conflicting results gathered from these kinds of trials .
overall , our results indicate that the mthfr 677tt genotype , along with homocysteine , bmi , and hdl - c levels , may help to identify apparently healthy females at increased risk for diabetes .
menon et al reported that hyperhomocysteinemia did not appear to be a risk factor for all - cause or cvd mortality , and prior studies demonstrating an association between homocysteine and cvd risk may have inadequately adjusted for the confounding effects of kidney function .
we also observed that mildly decreased egfr ( < 90  ml / min/1.73  m ) , but not homocysteine , was associated with the risk of new - onset disease .
nevertheless , more studies are needed to verify if our findings can be generalized to other populations or ethnicities , particularly individuals with lower egfr levels or obvious chronic kidney disease .
the strengths of our study include the 7-year prospective follow - up of middle - aged rural chinese men and women , and the comprehensive adjustments for the major traditional risk factors for nod , including baseline fpg and ir .
first , we did not measure glycosylated hemoglobin levels or perform glucose - tolerance tests .
second , we did not have information regarding family history of diabetes . however , further adjustment for family history of diabetes did not change the significant association between mthfr 677ct polymorphism and prevalence of diabetes in our previous study .
lastly , previous studies have shown that nonalcoholic fatty liver disease ( nafld ) was strongly associated with ir and diabetes .
unfortunately , we were not able to examine this effect in the current study due to the lack of data on nafld .
, we found that individuals with mthfr 677tt genotype had a significantly higher risk of nod among females , particularly in those with higher bmi or lower hdl - c levels .
the higher risk of nod associated with mildly decreased egfr ( < 90  ml / min/1.73  m ) also warrants more investigation .
our study findings , if further confirmed , will provide new strategies to identify apparently healthy population at increased risk for diabetes .
furthermore , considering the higher frequency of mthfr 677tt genotype in china and the higher stroke risk associated with tt genotype , our results also provide some explanations for the ethnic differences in diabetic complications seen between east asian patients and europeans .
 abstracteast asian patients with diabetes have a higher risk for renal complications and strokes than europeans . 
 we aimed to evaluate the effect of methylenetetrahydrofolate reductase ( mthfr ) gene 677ct polymorphism , which was associated with a higher stroke risk and was common in the chinese population , as well as homocysteine and estimated glomerular filtration rate ( egfr ) levels on the risk of new - onset diabetes ( nod).a total of 2422 subjects without diabetes were followed - up for 7 years . 
 nod was defined as fasting plasma glucose 7.0  mmol / l or self - reported physician diagnosis of diabetes.compared with subjects with mthfr 677cc genotype , those with tt genotype had a higher risk of nod in females ( odds ratio 2.78 , 95% confidence interval 1.395.56 ) but not in males ( 0.80 , 0.401.61 , p for interaction  =  0.008 ) . 
 furthermore , mthfr 677ct polymorphism was more strongly associated with the risk of nod among females with higher body mass index ( bmi , 23 vs < 23  kg / m2 , p for interaction  =  0.009 ) or lower high - density lipoprotein - cholesterol ( hdl - c , < 1.3 vs 1.3  mmol / l , p for interaction  =  0.015 ) levels . hyperhomocysteinemia ( 16 vs < 10  
 mol / l ) was not significantly associated with nod in males ( 0.88 , 0.421.85 ) or females ( 1.52 , 0.653.57 ) . 
 however , mildly decreased egfr ( < 90 vs 90120  ml / min/1.73  m2 ) was associated with nod mainly in males ( 1.96 , 1.013.78 ; females , 0.74 , 0.321.72 , p for interaction  =  0.134).females with mthfr 677tt genotype had a significantly higher risk of nod , particularly those with higher bmi or low hdl - c levels . the higher risk of nod associated with mildly decreased egfr also warrants more investigation . 
 our results provide insights into the ethnic differences of diabetic complications between east asian patients and europeans . 