Predictive value of folate, vitamin B12 and homocysteine levels in late-life depression

Background

The role of folate, vitamin B₁₂ and homocysteine levels in depressionis not clear.

Aims

To investigate cross-sectional and prospective associationsbetween folate, B₁₂ and homocysteine levels and late-life depression.

Method

A total of 732 Korean people aged 65 years or over were evaluatedat baseline. Of the 631 persons who were not depressed, 521(83%) were followed over a period of 2–3 years and incidentdepression was ascertained with the Geriatric Mental Stateschedule. Serum folate, serum vitamin B₁₂ and plasma homocysteinelevels were assayed at both baseline and follow-up.

Results

Lower levels of folate and vitamin B₁₂ and higher homocysteine levels at baseline were associated with a higher risk of incidentdepression at follow-up. Incident depression was associatedwith a decline in vitamin B₁₂ and an increase in homocysteinelevels over the follow-up period.

Conclusions

Lower folate, lower vitamin B₁₂ and raised homocysteine levels may be risk factors for late-life depression.

INTRODUCTION

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INTRODUCTION

Folate, vitamin B₁₂, homocysteine and methylenetetrahydrofolate reductase (MTHFR) are involved in one-carbon transfer (methylation)reactions necessary for the production of monoamine neurotransmitters,phospholipids and nucleotides. Folate and vitamin B₁₂ deficiency, hyperhomocysteinaemia and the T677 allele of the MTHFR gene,which cause impaired methylation reactions in the central nervoussystem, have been associated with depressive disorders.^1,2 However not all studies have found such associations.^3,4 Discrepantfindings in previous studies may relate to their cross-sectional design. In particular, changes in appetite and diet associatedwith depressive states may affect nutritional status, so thatthe direction of cause and effect remains unclear. To addressthis limitation, we analysed data from a 2-year longitudinalstudy to investigate both cross-sectional and prospective associationsbetween these factors and depression in late life.

Method

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Method

A secondary analysis was made of data from a community-basedprospective survey of late-life psychiatric morbidity conductedin Kwangju, Republic of Korea, from 2001 to 2003, in collaborationwith the 10/66 International Dementia Research Program in Developing Countries.⁵ All participants gave written, formal informedconsent at each examination. This study was approved by theChonnam National University Hospital institutional review board.

Baseline sample and measurements
A cross-sectional survey of a geographically defined populationwas carried out in 2001. The sampling procedure and measurementshave been described previously.⁶ In brief, 732 community residentsaged 65 years or over within two catchment areas of Kwangjuwere recruited from national residents' registration lists (5%refusal rate). Examinations included a fully structured diagnostic interview for depression; blood samples taken for folate, vitamin B₁₂, homocysteine and MTHFR genotype; and formal assessmentof potential confounding factors.

Depression
Depression was assessed using the community version of the GeriatricMental State (GMS) schedule.⁷ This is a fully structureddiagnostic instrument in wide international use with an accompanyingcomputerised algorithm which provides likelihoods of individual diagnoses on a scale of 0 to 5. The GMS was translated intoKorean according to a formal standardisation process.⁸ Asin other studies, a `stage one' (non-hierarchical) confidencelevel of 3 or above from the Automated Geriatric Examinationfor Computer Assisted Taxonomy (AGECAT) algorithm was usedto define depression. The instrument and algorithm are designedto define current depression present at a level of severity warranting clinical intervention, and focus on the month precedingthe interview. Case-level depression encompasses both moderateand severe symptoms, and is therefore a broader syndrome thanDSM–IV major depression. The 0–5 AGECAT confidencescore at baseline was included as a covariate in secondaryanalysis to allow exploratory adjustment for depressive symptomseverity as an ordinal rather than a binary measure.

Blood samples and biochemical analyses
Blood samples were collected from the participants in a fastingstate and were taken in the morning where possible. The sampleswere drawn into tubes of ethylenediamine tetra-acetic acid(EDTA), centrifuged, separated into plasma aliquots and storedat –70°C within 2 h of collection. Biochemical assayswere carried out after 3 years. Serum folate and vitamin B₁₂ levels were determined using an immunoassay, and total plasmahomocysteine level was measured by high-performance liquidchromatography. The MTHFR C677T genotype was determined bya polymerase chain reaction (PCR) and HinFI restriction enzymedigestion as described previously,⁹ with minor modification:HinFI digestion (1.5 U per 25 µl reaction mixture) wasperformed directly in the PCR tube at 37°C for 4 h before analysis of restriction fragments by polyacrylamide gel electrophoresis. Allele frequencies were estimated by gene counting and observednumbers of each genotype were compared with those expectedunder Hardy–Weinberg equilibrium.

Other measurements
Age, gender and education of the participants were recorded.Cognitive function was evaluated by the Korean version of theMini-Mental State Examination (MMSE).¹⁰ Disability was assessedby means of the Korean version of the World Health OrganizationDisability Assessment Schedule II (WHODAS–II).¹¹ Smokinghistory and current smoking status were ascertained. A lifetime history of alcohol consumption was obtained from the participants,and corroboration from family members was sought. Problem drinkingwas defined on the basis of consumption over the previous 3months of more than 14 alcoholic drinks per week for men ormore than 7 drinks per week for women, in accordance with guidelinesfrom the National Institute of Alcohol Abuse and Alcoholism.¹²Daily physical activity, taking into account both work andleisure activity, was ascertained and sedentary lifestyle wasdefined as a binary variable. For vascular risk factors anddisorders a summary `vascular risk' score was developed fromsumming self-reported disorders (stroke, heart disease, hypertension,diabetes), measured obesity (body mass index >25 kg/m²)and hypercholesterolaemia (fasting cholesterol >5.1 mmol).Serum creatinine level was also assayed, since impaired renalfunction may elevate serum metabolite levels independent ofvitamin intake.

Follow-up evaluation
Follow-up was carried out in 2003.¹³ The mean follow-up periodwas 2.4 years (s.d.=0.3). Attempts were made to follow up all previous participants. Identical procedures were used to identifydepression (GMS–AGECAT) and further blood samples forfolate, vitamin B₁₂ and homocysteine were collected, centrifugedwithin 1 h and stored at –70°C. Assays were doneafter 1 year. Vitamin supplementation was investigated in thecontext of an inventory taken of all prescription and non-prescriptionmedication taken in the past month.

Statistical analysis
Statistical analyses were carried out using SPSS version 12.0for Windows. Associations between baseline depression and baselinequintiles of folate, vitamin B₁₂ and homocysteine levels wereassessed by ${chi}$ ²-tests (linear trend). Associations with demographic characteristics, assessment scales (MMSE and WHODAS–II),lifestyle characteristics (smoking, problem drinking and physicalactivity), vascular risk or disease and serum creatinine levelwere investigated using t-, ${chi}$ ²- or Mann–Whitney U-testsas appropriate. Odds ratios and their 95% confidence intervalswere calculated for associations between baseline depressionand baseline quintiles of folate, vitamin B₁₂ and homocysteine,and for MTHFR genotype, in logistic regression models afteradjustment for the other independent variables. For all analyses,quintiles of folate, vitamin B₁₂ and homocysteine were enteredas ordinal variables with one degree of freedom, in accordance with an a priori hypothesis that that associations, if present,would show linearity across the distributions.

For investigating prospective associations, participants withcase-level depression at baseline were excluded, and case-leveldepression at follow-up (incident depression) was treated asthe dependent variable. Associations between incident depressionand baseline quintiles of folate, vitamin B₁₂ and homocysteinewere estimated in logistic regression models both before andafter adjustment for relevant factors including vitamin supplementation.In a secondary analysis, associations between baseline folate,vitamin B₁₂ and homocysteine levels and depression at follow-upwere recalculated for the total followed-up sample and thenfurther adjusted for baseline depression scale score (AGECAT0–5 scale). Further exploratory analyses for the incidentdepression analysis were carried out to investigate effectmodification of the three exposure–outcome associationsby gender and MTHFR genotype, particularly in view of previous findings that the impact of folate deficiency may be modifiedin this respect.^1,14

In participants without depression at baseline, change in levelsof folate, vitamin B₁₂ and homocysteine over the follow-upwere calculated and re-categorised by quintiles. Associationsbetween changes in these levels and incident depression werecalculated and investigated further in identical logistic regressionmodels to the analyses of baseline levels as predictors.

Final analyses were carried out to investigate associationswith standard categories of folate and vitamin B₁₂ deficiencyand hyperhomocysteinaemia.^4,15 Folate deficiency was definedon the basis of levels below 11.4 nmol/l and a homocysteinelevel higher than 13.9 µmol/l; vitamin B₁₂ deficiencyas a level less than 258 pmol/l; and hyperhomocysteinaemia asa plasma level above 15.0 µmol/l.

Results

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Results

Participants' characteristics at baseline
Of 732 participants at baseline, case-level depression was presentin 101 (13.8%). Mean levels of folate, vitamin B₁₂ and homocysteinefor the total sample were 24.4 nmol/l (s.d.=12.5), 385.6 pmol/l(s.d.=168.3) and 12.8 µmol/l (s.d.=5.7) respectively.Frequencies of the MTHFR allele were C 0.45 and T 0.55, andthe genotype distribution was C/C 18.7%, C/T 52.7% and T/T28.6% (test for Hardy–Weinberg equilibrium: ${chi}$ ²=2.091, P>0.05).Folate level was correlated positively with vitamin B₁₂ level(r=0.112, P=0.002) and negatively with homocysteine level (r=–0.310,P<0.001). Vitamin B₁₂ level was negatively correlated withhomocysteine level (r=–0.289, P<0.001). Homocysteinelevels were significantly associated with MTHFR genotype, withmean levels of 12.1 µmol/l (s.d.=5.6), 12.4 µmol/l(s.d.=4.6) and 13.8 µmol/l (s.d.=7.2) for the C/C, C/Tand T/T genotypes respectively (F=5.301, P=0.005). There wasno association between MHTFR genotype and folate or vitaminB₁₂ levels (all P values >0.1). Other characteristics ofthe sample and unadjusted associations with depression at baselineare summarised in Table 1.

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Table 1 Baseline characteristics of the study sample

Of 631 participants without depression at baseline, 521 (83%)completed all evaluations at follow-up and formed the studysample. Of the remaining 110, contact could not be establishedwith 58 (52%), 23 (21%) had died, 21 (19%) refused to participateand 8 (7%) were too unwell. Baseline characteristics of participantsfrom the baseline non-depressed group who were followed up are displayed in the last column of Table 1. Between the participantsand non-participants at follow-up, there was no substantialdifference in any independent variable (all P values >0.06).Mean changes in levels from baseline to follow-up were as follows:folate –4.9 nmol/l (s.d.=12.1), vitamin B₁₂ +48.0 pmol/l(s.d.= 139.7) and homocysteine +1.6 µmol/l (s.d.=5.0). Figure 1 summarises the prevalence and incidence of depressionaccording to baseline levels of folate, vitamin B₁₂ and homocysteine,and change in these levels over the follow-up period.

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Fig. 1 Rates of prevalence (a) and incidence (b) of depression according to baseline levels of folate, vitamin B₁₂ and homocysteine, and change in these levels over a 2-year follow-up period (c).

Baseline folate, vitamin B₁₂ and homocysteine levels, and baseline depression
Depression at baseline was associated with lower levels of vitamin B₁₂ ( ${chi}$ ²=4.190, P=0.041) and higher levels of homocysteine ( ${chi}$ ²=4.901,P=0.027), but was not significantly associated with folatelevels ( ${chi}$ ²=1.443, P=0.230) (Fig. 1). These findings persistedafter adjustment for potential confounders (Table 2). Prevalenceof depression by MTHFR genotype was 14.6% for C/C, 15.0% forC/T and 11.0% for T/T ( ${chi}$ ²=1.191, P=0.275).

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Table 2 Logistic regression models for the association between baseline folate, vitamin B₁₂ and homocysteine levels and baseline depression (n=732)

Baseline folate, vitamin B₁₂ and homocysteine levels, and incident depression
Incident depression was associated with lower baseline levelsof folate ( ${chi}$ ²=6.701, P=0.010) and vitamin B₁₂ ( ${chi}$ ²=6.317, P=0.012)and higher baseline levels of homocysteine ( ${chi}$ ²=5.335, P=0.021) (Fig. 1). Adjusted associations between these factors aredisplayed in Table 3. In summary, incident depression wasassociated with all three factors in the directions anticipated,with associations remaining significant after adjustment for other covariates (Table 3, model 6). When the three bloodlevels of interest were entered in combination (Table 3, models7–9) the associations with lower folate and vitamin B₁₂were reduced only marginally when adjusted for each other,with larger reductions when adjusted for homocysteine. On theother hand, the association between raised baseline homocysteinelevel and incident depression was reduced substantially when adjusted for individual vitamin levels. Incident depressionwas not associated with MTHFR genotype ( ${chi}$ ²=2.346, P=0.167).In a secondary analysis of the whole followed sample, the associationsbetween folate, vitamin B₁₂ and homocysteine, and depressionat follow-up were not substantially changed when adjusted forbaseline depression scale score (unadjusted odds ratios 1.24,1.28 and 1.18 respectively, adjusted odds ratios 1.23, 1.27and 1.18 respectively). In further exploratory stratified analyses,the association between descending folate and incident depression was significantly modified by MTHFR genotype: odds ratios fordecreasing folate quintiles were 1.18 (95% CI 0.79–1.76),1.22 (95 CI 0.86–1.73) and 1.85 (95% CI 1.14–3.00)within CC, CT and TT genotypes respectively (P=0.021 for statisticalinteraction). No significant interaction was found betweenMTHFR genotype and vitamin B₁₂ or homocysteine as exposures,no significant gender interaction was found for any exposureand no significant two-way or three-way interactions were foundbetween the three exposures of interest in predicting incidentdepression (data not shown). Among the 732 participants at baseline, a previous history of depression prior to age 60 years wasreported by 16 (16%) of the 101 participants with current depressionand by 17 (3%) of the remaining 631 participants. The findingsof interest were not materially altered following restrictionto those without a history of depression.

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Table 3 Logistic regression models for the association between baseline folate, vitamin B₁₂ and homocysteine levels, and incident depression over the 2-year follow-up period (n=521)

Incident depression and co-occurring changes in folate, vitamin B₁₂ and homocysteine levels
Incident depression was more frequent in people with a relativedecline in vitamin B₁₂ levels ( ${chi}$ ²=5.735, P=0.017) and witha relative increase in homocysteine ( ${chi}$ ²=6.594, P=0.010) (Fig. 1), whereas no association was found with change in folate levels ( ${chi}$ ²=0.971, P=0.324). Adjusted associations between these factorsare displayed in Table 4. The association between decline invitamin B₁₂ levels and incident depression remained strongafter adjustment for other covariates, was increased in strengthafter adjustment for vitamin supplementation at follow-up,and was decreased in strength following adjustment for homocysteinechange. The association between an increase in homocysteinelevels and incident depression changed little following adjustmentfor all other covariates.

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Table 4 Logistic regression models for the association between change in folate, vitamin B₁₂ and homocysteine levels, and incident depression (n=521)

Associations with clinical categories of folate and vitamin B₁₂ deficiency and hyperhomocysteinaemia
The prevalence of baseline folate deficiency was 4.0%, vitamin B₁₂ deficiency 16.8% and hyperhomocysteinaemia 22.1%. Oddsratios for associations with baseline depression were 1.32(95% CI 0.49–3.54) for folate deficiency, 1.57 (95% CI0.94–2.61) for vitamin B₁₂ deficiency and 1.78 (95% CI1.13–2.84) for hyperhomocysteinaemia. After adjustmentfor the other factors listed in Table 2, respective odds ratios were 1.86 (95% CI 0.59–5.80), 1.91 (95% CI 1.08–3.39)and 1.78 (95% CI 1.03–3.08). Respective odds ratios forincident depression adjusted for other covariates listed in Table 3 (model 6) were 1.94 (95% CI 0.58–6.47), 1.78(95% CI 0.90–3.51) and 1.69 (95% CI 0.88–3.26).

Discussion

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Discussion

To our knowledge, this study is the first community-based, prospective investigation of associations between folate, vitamin B₁₂, homocysteine and late-life depression. Principal findings werethat incident depression was predicted by lower folate andvitamin B₁₂ levels and higher homocysteine levels 2 years previously,and was associated with a decline in vitamin B₁₂ levels andan increase in homocysteine levels over the intervening period.No direct association with MTHFR genotype was found, althoughthe associations between folate levels and incident depressionwere modified by this factor. Associations between higher baseline homocysteine levels and incident depression were partly accountedfor by vitamin B₁₂ and folate levels.

Methodological issues
Previous community studies investigating the association betweenthese factors and depression have been cross-sectional in design.^1,4,15–19 This limits the extent to which causal relationships can beclarified, since measures of nutritional status such as vitaminlevels may be affected by the emergence of depressed statesand associated alterations in appetite and food intake. Relativedeficiency may, in turn, account for associations with raised homocysteine levels. Most studies have also been limited inthe use of brief screening instruments to define depression,^1,15–19 and in numbers of potential confounding factors considered,or in the specific nature of the cohorts analysed. Strengthsof our study were that prospective data on both depressionand the blood assays of interest were obtained from a communitypopulation, that depression was ascertained using a widely validated diagnostic schedule, and that a large number of potential confoundingfactors were considered in the analyses. The follow-up ratewas reasonable and not apparently differential with respectto risk factors of interest. The study sample was restrictedto older age ranges, but it is this group who are likely tobe most vulnerable to nutritional deficiency. Limitations ofthe study were that data on vitamin supplementation were notavailable at the baseline evaluation, and that at the follow-upevaluation the information on mental health was restrictedto the previous month. Detailed constituents of vitamin preparationswere also not available. The statistical models were constructed with the a priori assumption of linear associations betweenthe three exposures and depression outcomes. Figure 1 indicatesthat this may not be a completely appropriate assumption. However,in the absence of obvious mechanistic explanations for non-linearity,the models were not changed.

The nature of the outcome should also be considered. Most `case' participants will have had moderate levels of depression andthe results may not be generalisable to secondary care clinicalsamples with more severe syndromes. Furthermore, the outcomewas restricted to a single composite `diagnosis', without specificanalyses of clinical sub-types, comorbidity or particular symptomprofiles. Finally, a prospective analysis was used treating depression as an `incident' outcome which is unlikely to reflectfully complex symptom and syndrome trajectories (and whichmay have missed clinical episodes occurring and then recoveringbetween the examination points). Depression at baseline wasexcluded as a binary variable; however, a secondary analysis carried out for the whole followed sample did not suggest asubstantial contribution of syndrome prominence at baselinein accounting for the associations of interest.

Folate deficiency and depression
Previous case–control studies using clinical samples havereported significant associations between folate deficiencyand prevalence, severity and duration of depressive disorders.²⁰This has been replicated in some community studies,¹⁸ butnot in others.^1,4,15 These discrepant results might be dueto differences in sample characteristics, depression ascertainmentor blood assays. In our study folate deficiency was not associatedwith depression in cross-sectional analyses, but lower folatelevels were associated with a higher likelihood of incident depression 2 years later. The cross-sectional association betweendepression and folate deficiency might be obscured by selectionbias if people with both depression and nutritional deficiencywere less likely to participate, or if they were more proneto be hospitalised and therefore underrepresented in communitysamples. It is also possible that people with longer-lasting depressive states, who are over-represented in cross-sectionalsurveys, may regulate their diet in a way that might compensatefor earlier deficiencies. The prevalence of folate deficiencyat baseline was relatively low, but this is likely to be explainedby the relatively high intake of folate-containing green vegetablesin Korean populations, which has been previously recognised.²¹ Nevertheless, folate level remained negatively correlated withhomocysteine level in our sample, as has been reported elsewhere.¹The lower prevalence of folate deficiency might have obscuredthe association between the folate deficiency and depressionat baseline in this particular population. The prospectiveassociation between lower folate levels and incident depressionwas not explained by other potential confounding factors (Table 3),and homocysteine, as a potential mediating factor, explainedonly a small proportion of this association. It is of interestthat the association between lower folate and incident depressionwas significantly modified by MTHFR genotype, with strongestassociations in those with the T/T genotype. A recent study suggested that, since the MTHFR gene influences the functioningof the folate metabolic pathway, folate or its derivativesmight be causally related to risk of depression.¹⁴

Vitamin B₁₂ deficiency and depression
The cross-sectional significant association observed betweenlower vitamin B₁₂ levels and depression is consistent withprevious findings from both clinical samples²² and community populations,^4,15 although not all studies have found this.¹In prospective analyses, incident depression was associatedboth with lower baseline vitamin B₁₂ levels and with a previousdecline in vitamin B₁₂ levels from baseline to follow-up. VitaminB₁₂ is required for the synthesis of S-adenosylmethionine,which is an important methyl donor in many important methylationreactions in the central nervous system. Inhibited synthesisof S-adenosylmethionine may reduce monoamine neurotransmittersynthesis, and S-adenosylmethionine has been suggested to haveantidepressant activity.²³ A causal relationship between vitaminB₁₂ levels and depression is supported by the prospective findings.Cross-sectional associations may also be explained by a depressivestate adversely influencing dietary intake and resulting inlower circulating vitamin B₁₂ levels. This is supported bythe association between vitamin B₁₂ decline and incident depression.However, inferences can only be tentative since the temporalrelationship between vitamin B₁₂ decline and affective statecould not be established within the follow-up period. An important limitation with most research, including this study, is thatcirculating vitamin B₁₂ is only a proxy marker of cobalamindeficiency at a cellular level. Methylmalonic acid is a morespecific marker of functional vitamin B₁₂ status,²⁴ but was not assayed in this study.

Hyperhomocysteinaemia and depression
Higher homocysteine levels have been associated with depressivesymptoms in both middle-aged and older community populations.^1,17,19 Our findings were similar. Higher homocysteine levels havebeen found to be associated with disability²⁵ and with cerebrovascular diseases,²⁶ which are themselves potential risk factors fordepression. However, we found little evidence of confoundingby these factors to the extent to which they were measuredin the study. Raised homocysteine levels have also been associated with cognitive impairment,²⁷ which may be a potential confounder.However, although in the baseline sample raised homocysteinelevel was associated with lower MMSE score (data not shown), neither cross-sectional nor prospective associations between hyperhomocysteinaemia and depression were altered followingadjustment for this. Incident depression was associated witha previous rise in homocysteine levels. This may indicate aneffect of a depressive state. Although changes in vitamin B₁₂or folate levels did not appear to account for this association,these factors might not have been sufficiently accurate markers of bioavailability, limiting the inferences that can be drawn.

Interactions between folate, vitamin B₁₂ and homocysteine levels
The correlation coefficients between the levels of folate, vitamin B₁₂ and homocysteine were significant but only modest in strength, and lower than those found in previous studies.¹ In addition,there was no significant interaction between the folate, vitamin B₁₂ and homocysteine levels on the prevalent or incident depression.Associations between baseline folate and vitamin B₁₂ levelsand incident depression were in part accounted for by homocysteine levels, suggesting that homocysteine might be a causal pathwayfactor between nutritional status and depression. The effectof adjusting the homocysteine–depression associationfor folate or vitamin B₁₂ should, however, be viewed with caution,since the circulating levels assayed are only proxy markersfor their function at a cellular level and confounding effectsmay be underestimated.

MTHFR genotype and depression
A direct association between MTHFR genotype and depression wasnot supported. This result was consistent with one Japanesecase–control study²⁸ and an Australian community study,³although an association between MTHFR T/T homozygosity anddepression was found in another Japanese case–control study²⁹ and in a Norwegian community study.¹ MTHFR T/T genotypefrequency in the sample reported here was 29%, higher than thatin the Japanese samples (13–14%) and in those from Australiaand Norway (12% and 8% respectively). In our study there wassome evidence from an exploratory analysis that the T/T genotypemight modify the association between low folate and depression.An overall association between MTHFR genotype and depressionmight conceivably have been reduced because of the relativelylow prevalence of folate deficiency in this sample (due to the traditional Korean vegetable-rich diet).

Implications for public health and future research
Our findings in this prospective community study support rolesfor folate, vitamin B₁₂ and homocysteine levels in the aetiologyof late-life depression. From a public health perspective,there may be good arguments for focusing interventions forthe prevention of depression on nutritionally deficient, frailpopulations. Although the use of vitamin supplements did not substantially modify the observed associations, further researchis likely to be required as the ascertainment in this studymight have been incomplete and obscured by dietary habits.Relationships with the dose, duration and (particularly) constituentsof vitamin supplements should be investigated. However, itshould be borne in mind that the results of observational research are often not confirmed by interventional studies. For example,a recent study reported that homocysteine reduction with Bvitamins did not reduce the risk of recurrent cardiovasculardisease after acute myocardial infarction,³⁰ despite the factthat raised homocysteine levels had repeatedly been found to be associated with increased risk of cardiovascular diseasein observational studies. In addition, although a role of MTHFRgenotype was not supported in our study, gene–environmentand gene–gene interactions require further evaluation.

ACKNOWLEDGMENTS

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ACKNOWLEDGMENTS

The study was funded by a grant from the Korea Health 21 Researchand Development Project, Ministry of Health and Welfare, Republicof Korea (A050047).

REFERENCES

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