Intrapair Differences Research Articles

Characterising metabolically healthy obesity in weight-discordant monozygotic twins

Not all obese individuals display the metabolic disturbances commonly associated with excess fat accumulation. Mechanisms maintaining this 'metabolically healthy obesity' (MHO) are as yet unknown. We aimed to study different fat depots and transcriptional pathways in subcutaneous adipose tissue (SAT) as related to the MHO phenomenon. Sixteen rare young adult obesity-discordant monozygotic (MZ) twin pairs (intra-pair difference (∆) in BMI ≥ 3 kg/m(2)), aged 22.8-35.8 years, were examined for detailed characteristics of metabolic health (subcutaneous, intra-abdominal and liver fat [magnetic resonance imaging/spectroscopy]), OGTT, lipids, adipokines and C-reactive protein (CRP). Affymetrix U133 Plus 2.0 chips were used to analyse transcriptomics pathways related to mitochondrial function and inflammation in SAT. Based on liver fat accumulation, two metabolically different subgroups emerged. In half (8/16) of the pairs (∆weight 17.1 ± 2.0 kg), the obese co-twin had significantly higher liver fat (∆718%), 78% increase in AUC insulin during OGTT and CRP, significantly more disturbance in the lipid profile and greater tendency for hypertension compared with the lean co-twin. In these obese co-twins, SAT expression of mitochondrial oxidative phosphorylation, branched-chain amino acid catabolism, fatty acid oxidation and adipocyte differentiation pathways were downregulated and chronic inflammation upregulated. In the other eight pairs (∆weight 17.4 ± 2.8 kg), the obese co-twin did not differ from the non-obese co-twin in liver fat (∆8%), insulin sensitivity, CRP, lipids, blood pressure or SAT transcriptomics. Our results suggest that maintenance of high mitochondrial transcription and lack of inflammation in SAT are associated with low liver fat and MHO.

Hereditary Factors and Tooth Dimensions: A Twin Study

Introduction : Determination of the actual level of genetic determinance of occlusal variation could have a significant effect on prognosis and treatment planning. Aim: The objective of this study was to evaluate the hereditary variations occurring among the members within monozygotic and dizygotic twin pairs based on widest mesiodistal measurements in relation to the posterior permanent dentition. Materials and methods: Twenty-eight adult twin pairs (16 monozygotic pairs, 12 dizygotic pairs) in the age group of 14 to 55 years were used for the study. Mesiodistal measurements of maxillary and mandibular permanent posterior teeth at their maximum width were considered. Intrapair differences were calculated, the differences in the two groups were compared and the level of significance was tested. Results: Significant differences between the twin groups was found in relation to all the teeth studied except the maxillary and mandibular second molars. Conclusion: Genetically conditioned variations of highly significant nature were seen in 12 of the 16 posterior teeth studied, thereby confirming the evidence that size of teeth is under genetic control.

Genetic and environmental factors in associations between infant growth and adult cardiometabolic risk profile in twins

Accelerated infant growth is associated with an altered, mostly adverse adult cardiometabolic risk profile. The importance of genetic and environmental factors to these associations is unclear. The objective was to examine the importance of genetic and environmental factors in the associations between infant growth and adult cardiometabolic risk factors (anthropometric characteristics, lipids, insulin sensitivity, leptin, blood pressure, and fibrinogen) in twins. Cardiometabolic risk factors were assessed in 240 twin pairs (aged 18-34 y) from the East Flanders Prospective Twin Survey. Infant growth was defined as change in weight z score. We regressed intrapair differences in growth during 4 growth windows (0-1, 1-6, 6-12, and 12-24 mo) against intrapair differences in the risk factors in monozygotic and dizygotic twins separately. Within monozygotic twin pairs only, associations between infant growth and most adult lipids, glucose, leptin, and blood pressure (eg, systolic blood pressure: b = 5.95 mm Hg per change in z score, P = 0.01 in monozygotic twins; b = -1.64, P = 0.82 in dizygotic twins from 12 to 24 mo) were found. Within dizygotic twin pairs only, associations between growth and triglycerides and fibrinogen (eg, fibrinogen: b = 0.07 ln mg/dL per change in z score, P = 0.31 in monozygotic twins; b = 0.79, P = 0.01 in dizygotic twins from 0 to 1 mo) were identified. Most associations showed a detrimental effect of accelerated growth, but beneficial associations were also identified (eg, total-to-high-density-lipoprotein cholesterol ratio: b = -0.22 per change in z score from 1 to 6 mo, P = 0.008 in monozygotic twins). Our data showed that environmental factors play a role in the associations between infant growth and most adult lipids, glucose, leptin, and blood pressure, whereas genetic factors are involved regarding triglycerides and fibrinogen.

A Twin-Sibling Study on the Relationship Between Exercise Attitudes and Exercise Behavior

Social cognitive models of health behavior propose that individual differences in leisure time exercise behavior are influenced by the attitudes towards exercise. At the same time, large scale twin-family studies show a significant influence of genetic factors on regular exercise behavior. This twin-sibling study aimed to unite these findings by demonstrating that exercise attitudes can be heritable themselves. Secondly, the genetic and environmental cross-trait correlations and the monozygotic (MZ) twin intrapair differences model were used to test whether the association between exercise attitudes and exercise behavior can be causal. Survey data were obtained from 5,095 twins and siblings (18-50 years). A genetic contribution was found for exercise behavior (50 % in males, 43 % in females) and for the six exercise attitude components derived from principal component analysis: perceived benefits (21, 27 %), lack of skills, support and/or resources (45, 48 %), time constraints (25, 30 %), lack of energy (34, 44 %), lack of enjoyment (47, 44 %), and embarrassment (42, 49 %). These components were predictive of leisure time exercise behavior (R(2) = 28 %). Bivariate modeling further showed that all the genetic (0.36 < |rA| < 0.80) and all but two unique environmental (0.00 < |rE| < 0.27) correlations between exercise attitudes and exercise behavior were significantly different from zero, which is a necessary condition for the existence of a causal effect driving the association. The correlations between the MZ twins' difference scores were in line with this finding. It is concluded that exercise attitudes and exercise behavior are heritable, that attitudes and behavior are partly correlated through pleiotropic genetic effects, but that the data are compatible with a causal association between exercise attitudes and behavior.

Serotonin Transporter Gene

The serotonin transporter gene has been hypothesized to influence, possibly in interaction with environmental factors, the vulnerability for depression. So far, genetic studies have tested the association of the repeat polymorphism (5-HTTLPR) with depression and whether it is moderated by exposure to stressful events. This has not yielded unequivocal results, even across meta-analyses. However, environmental factors may induce epigenetic changes in the structure of DNA that can influence gene expression. These epigenetic effects may be independent of the genetic polymorphisms in the gene region. This editorial reviews an article in this issue that compared the intrapair differences in depressive symptoms in monozygotic twin pairs with the intrapair differences of methylation at cytosine-guanine dinucleotide sites in the promoter region of the serotonin transporter gene. Differences in depressive symptoms were correlated with differences in methylation status, such that higher methylation, which, in this sample of identical twins, must be environmental in origin, is associated with more depressive symptoms. Noteworthy is the fact that the epigenetic effects were independent of the 5-HTTLPR. These results should encourage genome-wide testing of the contribution of epigenetic effects to depression.

Association Between Promoter Methylation of Serotonin Transporter Gene and Depressive Symptoms

Epigenetic mechanisms have been implicated in the pathogenesis of psychiatric disorders. The serotonin transporter gene (SLC6A4) is a key candidate gene for depression. We examined the association between SLC6A4 promoter methylation variation and depressive symptoms using 84 monozygotic twin pairs. DNA methylation level in the SLC6A4 promoter region was quantified by bisulfite pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. The number of current depressive symptoms was assessed using the Beck Depressive Inventory II (BDI-II). The association between methylation variation and depressive symptoms was examined using matched twin-pair analyses, adjusting for body mass index, smoking, physical activity, and alcohol consumption. Multiple testing was controlled by adjusted false discovery rate (q value). Intrapair difference in DNA methylation variation at 10 of the 20 studied CpG sites is significantly correlated with intrapair difference in BDI scores. Linear regression using intrapair differences demonstrates that intrapair difference in BDI score was significantly associated with intrapair differences in DNA methylation variation after adjusting for potential confounders and correction for multiple testing. On average, a 10% increase in the difference in mean DNA methylation level was associated with 4.4 increase in the difference in BDI score (95% confidence interval = 0.9-7.9, p = .01). This study provides evidence that variation in methylation level within the promoter region of the serotonin transporter gene is associated with variation in depressive symptoms in a large sample of monozygotic twin pairs. This relationship is not confounded by genetic and shared environment. The 5-HTTLPR genotype also does not modulate this association.

Association of body mass index with arterial stiffness and blood pressure components: A twin study

RationaleObesity, blood pressure and arterial stiffness are heritable traits interconnected to each other but their possible common genetic and environmental etiologies are unknown. MethodsWe studied 228 monozygotic and 150 dizygotic twin pairs aged 18–82 years from Italy, Hungary and the United States, of which 45 monozygotic and 38 dizygotic pairs were discordant for body mass index (BMI; intrapair difference (Δ) in BMI ≥ 3 kg/m2). Blood pressure components and arterial stiffness were measured by TensioMed Arteriograph. ResultsHypertension was more prevalent among obese than non-obese individuals (55% vs. 29%, p < 0.001). Age-, sex- and country-adjusted heritability estimates were high for hemodynamic measures (45%–58%) and BMI (78%). According to bivariate Cholesky decomposition, phenotypic correlations between BMI and blood pressure components (r = −0.15 to 0.24, p < 0.05) were largely explained by additive genetic factors (65%–77%) with the remaining explained by the unique environment. When controlling for genetic factors within all monozygotic pairs, ΔBMI was significantly correlated with Δbrachial systolic blood pressure (SBP) and diastolic blood pressure (DBP), Δmean arterial pressure, and Δaortic SBP (r = 0.15–0.17, p < 0.05). For the same measures, heavier co-twins of BMI-discordant monozygotic pairs had significantly higher values than their leaner counterparts (p < 0.05). ConclusionBlood pressure components are moderately correlated with BMI, largely because of shared genetic factors. However, for the association of BMI with brachial SBP and DBP, aortic SBP and mean arterial pressure, acquired, modifiable factors were also found to be important.

Birth Weight and Adult Bone Metabolism Are Unrelated: Results From Birth Weight–Discordant Monozygotic Twins

Low birth weight (BW) has been associated with poor bone health in adulthood. The aim of this study was to investigate the association between BW and bone mass and metabolism in adult BW-discordant monozygotic (MZ) twins. A total of 153 BW-extremely discordant MZ twin pairs were recruited from the Danish Twin Registry. Serum vitamin D (25-hydroxyvitamin D [25OHD]) and bone turnover markers (BTMs) amino-terminal propeptide of type I procollagen (P1NP), pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (1CTP), and cross-linked C-telopeptide (CTX) were quantified. Femoral neck (FN), total hip (TH), lumbar spine (LS), and whole-body (WB) bone mineral density (BMD) (ie, FN-BMD, TH-BMD, LS-BMD, and WB-BMD, respectively) were measured using dual-energy X-ray absorptiometry (DXA). Twins were studied as single individuals using regression analyses with or without adjustment for height, weight, age, sex, and intrapair correlation. Within-pair differences were assessed using Student's t test and fixed-regression models. BW was not associated with BTMs, LS-BMD, TH-BMD, FN-BMD, or WB-BMD, but BW was associated with WB-BMC, and WB-Area after adjustments. Compared to the co-twin, twins with the highest BW were heavier and taller in adulthood (mean differences ± SD): 3.0 ± 10.5 kg; 1.6 ± 2.6 cm; both p < 0.001). Within-pair analyses showed that LS-BMD, TH-BMD, and FN-BMD tended to be higher in twins with highest BW (for all: mean difference 0.01 ± 0.1 g/cm(2) ; p = 0.08, 0.05, and 0.10, respectively). No difference was observed after adjustment for adult body size. Intrapair differences in BW were not associated with differences in any of the biochemical parameters or BMD. Small differences between twins in BMD were explained by dissimilarities in body size. These results suggest that BW and adult bone metabolism are unrelated.

Regional gray matter reductions are associated with genetic liability for anxiety and depression: An MRI twin study

BackgroundThe influence of genetic and/or environmental factors on the volumetric brain changes observed in subjects affected by anxiety and depression disorders remains unclear. The current study aimed to investigate whether genetic and environmental liabilities make different contributions to abnormalities in gray matter volume (GMV) in anxiety and depression using a concordant and discordant MZ twin pairs design. MethodsFifty-three magnetic resonance imaging (3T) brain scans were obtained from monozygotic (MZ) twins concordant (6 pairs) and discordant (10 pairs) for lifetime anxiety and depression disorders and from healthy twins (21 subjects). We applied voxel-based morphometry to analyse GMV differences. Concordant affected twins were compared to healthy twins and within-pairs comparisons were performed in the discordant group. ResultsGMV reductions in bilateral fusiform gyrus and amygdala were observed in concordant affected twins for anxiety and depression compared to healthy twins. No intrapair differences were found in GMV between discordant affected twins and their healthy co-twins. LimitationsThe sample size was modest. This might explain why no intrapair differences were found in the discordant MZ twin group. ConclusionsAs concordant affected MZ twins are believed to have a particularly high genetic liability for the disorder, our findings suggest that fusiform gyrus and amygdala gray matter reductions are related to a genetic risk for anxiety and depression. Discrepancies in regard to brain abnormalities in anxiety and depression may be related to the admixture of patients with GMV abnormalities mainly accounted for by genetic factors with patients presenting GMV mainly accounted for by environmental factors.

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles

BackgroundLow birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes.ResultsOverall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences.ConclusionsSevere intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies.

Genome-wide analysis of DNA methylation differences in muscle and fat from monozygotic twins discordant for type 2 diabetes.

BackgroundMonozygotic twins discordant for type 2 diabetes constitute an ideal model to study environmental contributions to type 2 diabetic traits. We aimed to examine whether global DNA methylation differences exist in major glucose metabolic tissues from these twins.Methodology/Principal FindingsSkeletal muscle (n = 11 pairs) and subcutaneous adipose tissue (n = 5 pairs) biopsies were collected from 53–80 year-old monozygotic twin pairs discordant for type 2 diabetes. DNA methylation was measured by microarrays at 26,850 cytosine-guanine dinucleotide (CpG) sites in the promoters of 14,279 genes. Bisulfite sequencing was applied to validate array data and to quantify methylation of intergenic repetitive DNA sequences. The overall intra-pair variation in DNA methylation was large in repetitive (LINE1, D4Z4 and NBL2) regions compared to gene promoters (standard deviation of intra-pair differences: 10% points vs. 4% points, P<0.001). Increased variation of LINE1 sequence methylation was associated with more phenotypic dissimilarity measured as body mass index (r = 0.77, P = 0.007) and 2-hour plasma glucose (r = 0.66, P = 0.03) whereas the variation in promoter methylation did not associate with phenotypic differences. Validated methylation changes were identified in the promoters of known type 2 diabetes-related genes, including PPARGC1A in muscle (13.9±6.2% vs. 9.0±4.5%, P = 0.03) and HNF4A in adipose tissue (75.2±3.8% vs. 70.5±3.7%, P<0.001) which had increased methylation in type 2 diabetic individuals. A hypothesis-free genome-wide exploration of differential methylation without correction for multiple testing identified 789 and 1,458 CpG sites in skeletal muscle and adipose tissue, respectively. These methylation changes only reached some percentage points, and few sites passed correction for multiple testing.Conclusions/SignificanceOur study suggests that likely acquired DNA methylation changes in skeletal muscle or adipose tissue gene promoters are quantitatively small between type 2 diabetic and non-diabetic twins. The importance of methylation changes in candidate genes such as PPARGC1A and HNF4A should be examined further by replication in larger samples.

A Genome-Wide Association Study of Monozygotic Twin-Pairs Suggests a Locus Related to Variability of Serum High-Density Lipoprotein Cholesterol

Genome-wide association analysis on monozygotic twin-pairs offers a route to discovery of gene environment interactions through testing for variability loci associated with sensitivity to individual environment/lifestyle. We present a genome-wide scan of loci associated with intra-pair differences in serum lipid and apolipoprotein levels. We report data for 1,720 monozygotic female twin-pairs from GenomEUtwin project with 2.5 million SNPs, imputed or genotyped, and measured serum lipid fractions for both twins. We found one locus associated with intra-pair differences in high-density lipoprotein cholesterol, rs2483058 in an intron of SRGAP2, where twins carrying the C allele are more sensitive to environmental factors(P=3.98 x 10-8). We followed up the association in further genotyped monozygotic twins (N= 1,261),which showed a moderate association for the variant (P= 0.200, same direction of an effect). In addition,we report a new association on the level of apolipoprotein A-ll (P= 4.03 x 1 o-8).

Adult glucose metabolism in extremely birthweight-discordant monozygotic twins

Low birthweight (BW) is associated with increased risk of type 2 diabetes. We compared glucose metabolism in adult BW-discordant monozygotic (MZ) twins, thereby controlling for genetic factors and rearing environment. Among 77,885 twins in the Danish Twin Registry, 155 of the most BW-discordant MZ twin pairs (median BW difference 0.5kg) were assessed using a 2h oral glucose tolerance test with sampling of plasma (p-)glucose, insulin, C-peptide, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. HOMA for beta cell function (HOMA-β) and insulin resistance (HOMA-IR), and also insulin sensitivity index (BIGTT-SI) and acute insulin response (BIGTT-AIR), were calculated. Subgroup analyses were performed in those with: (1) double verification of BW difference; (2) difference in BW >0.5kg; and (3) no overt metabolic disease (type 2 diabetes, hyperlipidaemia or thyroid disease). No intra-pair differences in p-glucose, insulin, C-peptide, incretin hormones, HOMA-β, HOMA-IR or BIGTT-SI were identified. p-Glucose at 120min was higher in the twins with the highest BW without metabolic disease, and BIGTT-AIR was higher in those with the highest BW although not in pairs with a BW difference of >0.5kg. BW-discordant MZ twins provide no evidence for a detrimental effect of low BW on glucose metabolism in adulthood once genetic factors and rearing environment are controlled for.

Circulating surfactant protein D is associated to mortality in elderly women: A twin study

BackgroundSurfactant protein D (SP-D) is produced in the lungs and additional mucosal surfaces. Systemic SP-D levels are previously associated to aging-related- and lifestyle-related disorders and predicts mortality in cardiovascular and lung diseases. However, the association between higher serum SP-D levels and mortality in the general population is unknown. We hypothesized that increased systemic levels of SP-D may be used as prognostic factor for assessing the mortality in the elderly. Methods and resultsSP-D serum levels were measured in 689 elderly subjects and mortality ratios were investigated after a 13-year follow-up period. Survival analysis showed that increasing quartiles of serum SP-D levels were associated to mortality in 70+ year old women (hazard ratio [HR], 1.29; 95% confidence interval [CI], 0.93–1.78; p=0.032) adjusted for age, smoking and BMI. Women with SP-D levels above 2100ng/ml had significantly increased mortality when compared to elderly women with SP-D levels equal to or below 2100ng/ml (HR, 1.45; 95% CI, 1.12–1.88; p=0.005).The likelihood that the female twin with the highest SP-D level died first increased with increasing SP-D levels (p=0.031) – that is, the bigger intra-pair difference in SP-D level, the higher the probability that the twin with the highest measure died first (odds ratio [OR], 1.66; p=0.047). ConclusionThe study demonstrates that higher circulating SP-D levels are associated with increased mortality rate in elderly women in this population-based cohort study. SP-D may serve as a biomarker to track the cardio-pulmonary health status in elderly women.

Intrauterine growth characteristics of twins and those twins discordant birthweight

To investigate the intrauterine growth characteristics of twins and birthweight discordant twins (discordant twins). Total of 1010 twin pregnancies (2020 fetuses) with complete delivery records from the Department of Obstetrics and Gynecology, the First and Third Affiliated Hospital of SUN Yat-sen University between January 1, 2000 and July 31, 2010 were studied retrospectively. One handred and ninteen cases (238 fetuses) with intrapair birthweight difference ≥ 25% were determined as the discordant twins group, and the other 891 cases (1782 fetuses) with intrapair birthweight difference < 25% were identified as the concordant twins group. The singleton control group included 4042 singleton pregnancies in the same period. (1) Comparison of clinical data between the twins groups: the birthweight of larger-twin, smaller-twin and intrapair birthweight difference in the discordant twins group and the concordant twins group were (2090 ± 827) g, (1392 ± 592) g, (33.9 ± 9.3)%, and (2408 ± 543) g, (2191 ± 505) g, (8.9 ± 6.5)%, respectively, with significant differences (P < 0.01). The incidence of discordant twins was 11.78% (119/1010). Compared with the concordant twins group, the discordant twins group had higher proportion of monochorionic twins, and higher prevalence of pregnancy complications such as late miscarriage, abnormal umbilical insertion, twin-twin transfusion syndrome and hypertensive disorders in pregnancy (P < 0.05). (2) The characteristics of twin birthweight distribution: 1) In all the 2020 twins, 80.05% (1617/2020) fetuses had birthweight below the 50(th) percentile of the singleton control group, while 23.71% (479/2020) feeuses got birthweight below the 10(th) percentile of the singleton control group. 2) After 19(th) gestational week, the 50(th) and 90(th) percentile of all twins' birthweight were lower than those of singletons. After 38(th) gestational week, the birthweight of singletons kept increasing and reached its peak at 41(th) week, while the birthweight of twins reached its peak at 38(th) week, followed by a decline at 39 weeks, which was even lower than the 10(th) percentile of the singleton control group. 3) The distribution of birthweight of larger- and smaller-twin in the discordant twins group: 65 (54.6%, 65/119) larger-twins and one (0.8%, 1/119) smaller-twin had birthweight above the 50(th) percentile of all twins, while 5 (4.2%, 5/119) larger-twins and 97 (81.5%, 97/119) smaller-twins got birthweight below the 10(th) percentile of all twins. (1) The patterns of birthweight curves for each gestational week are different between twins and singletons. In order to evaluate the growth of twins, birthweight reference for twins should be employed. (2) According to the reference of twins birthweight, the most discordant twins are complicated with fetal growth restriction at least in one twin.

Abstract P194: Gene-Environment Interactions and Blood Lipid levels: a Twin Study

Genetic variation may affect blood lipid levels by directly determining levels (‘level genes’) and/or by influencing the response to environmental factors (‘variability genes’), i.e. gene-environment interaction. Previous dietary studies assessing variability gene effects have produced inconsistent results, perhaps due to a lack of statistical power, different dietary protocols, and/or differences in the age, sex, habitual diet, baseline lipid values and other genes of the participants. Monozygotic (MZ) twins can enhance our ability to identify variability genes because they are genetically identical and also share the same age, sex and aspects of their early environment. Therefore, all intrapair differences in blood lipid levels are due to environmental factors such as diet and lifestyle, and variation between genotypes in intrapair differences will indicate the existence of variability genes. We investigated the hypothesis that polymorphisms within the genes for apoB, apoE and lipoprotein lipase influence the blood lipid response to environmental factors. The subjects studied were 64 MZ male and female twin pairs aged 18-75 years who had been recruited for a study of coronary heart disease risk factors in twins. Blood samples already taken for analysis of fasting lipid levels were used for DNA extraction. Genotypes were determined for the polymorphisms apoB signal peptide insertion/deletion (I/D), apoB Xba I, apoE, and lipoprotein lipase Pvu II, Hind III and S447X. Mean intrapair differences in lipid levels adjusted for mean twin pair lipid level were calculated for each genotype group. In MZ twins, carriers of the apoB signal peptide D allele had smaller intrapair differences in total cholesterol (p=0.044) but larger intrapair differences in apoAI (p=0.035) than I/I twins, and apoE ε4 carriers had smaller intrapair differences in HDL-cholesterol than E3/3 and E3/2 twins (p=0.024). MZ twins with the Pvu II P-P- genotype had significantly greater intrapair differences in apoB than P-P+ or P+P+ twins. The Xba I, Hind III and S447X polymorphisms did not influence intrapair differences in lipid levels. In conclusion, the apoB signal peptide I/D, apoE and lipoprotein lipase Pvu II polymorphisms may act as variability genes and influence the susceptibility of blood lipid levels to dietary and pharmacological interventions for dyslipidaemia. The approach of using MZ twins to identify variability genes can be applied to other phenotypes, prior to determining the environmental factors that interact with the genes to influence the phenotype.

Abstract 049: Promoter Methylation of Glucocorticoid Receptor Gene Is Associated with Subclinical Cardiovascular Disease: a Monozygotic Twin Study

Background: Epigenetic mechanisms are increasingly being recognized as a key factor in the pathogenesis of cardiovascular disease (CVD). Endothelial dysfunction assessed by brachial artery flow-mediated dilation (FMD) is a marker for early atherosclerotic CVD, a predictor of future cardiovascular events. The glucocorticoid receptor gene (NR3C1) regulates a variety of biological processes including cardiovascular, metabolic, immunologic, and homeostatic functions. Genetic polymorphisms in NR3C1 have been associated with atherosclerosis and several cardiometabolic risk factors. However, no study has yet investigated the potential role of NR3C1 promoter methylation in atherosclerosis. Objective: To examine the potential association between NR3C1 promoter methylation and FMD in a monozygotic (MZ) twin sample. Method: We studied 84 male-male monozygotic twin pairs drawn from the Vietnam Era Twin Registry. These twin pairs were recruited by the Emory Twins Heart Study, which investigates psychological, behavioral and biological risk factors for subclinical cardiovascular disease using a twin design. The twins were free of a self-reported previous diagnosis of cardiovascular disease at the time of enrollment. Brachial artery FMD was measured by ultrasound. The methylation levels of 25 CpG residues in the NR3C1 promoter region in peripheral blood leukocytes was quantified by bisulfite pyrosequencing. The association between DNA methylation variation and FMD was examined using nonparametric regression by regressing the intra-pair difference in FMD on the intra-pair difference in DNA methylation level at each CpG site, separately, adjusting for potential confounders including age, smoking, body mass index (BMI), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), blood glucose level and systolic blood pressure. Multiple testing was controlled by the false discovery rate. Results: Mean methylation level of the 25 CpG sites encompassing the NR3C1 promoter region was 2.1%. Methylation levels at 7 of the 25 CpG residues were significantly associated with FMD. On average, a 10% increase in mean methylation was associated with a 0.13 increase in FMD (95% CI: 0.09–0.17; p &lt;0.0001) after controlling for confounding factors. Conclusion: Increased promoter methylation of NR3C1 is significantly associated with decreased risk for subclinical atherosclerosis assessed by FMD. Given the critical role of this gene in regulating hypothalamic-pituitary-adrenal axis (HPA) function, it is possible that aberrant promoter methylation of this gene may affect atherogenesis through its influence on the HPA system, a mechanism known to be involved in cardiovascular disease.

Epicardial Fat, Cardiac Dimensions, and Low-Grade Inflammation in Young Adult Monozygotic Twins Discordant for Obesity

Epicardial fat with its close proximity to coronary arteries has been suggested to be a significant predictor of cardiovascular disease. We studied the relations among acquired obesity, low-grade inflammation, and genetic factors in the accumulation of epicardial fat. A rare sample (n = 15) of healthy monozygotic (MZ) twin pairs discordant for obesity (intrapair difference in body mass index ≥3 kg/m(2)) and 9 concordant MZ pairs 23 to 33 years old were examined for cardiac structure, function, epicardial fat thickness (echocardiography), abdominal subcutaneous tissue, and visceral adipose tissue (VAT), liver fat (magnetic resonance imaging/spectroscopy), and serum high-sensitivity C-reactive protein. In the entire sample, MZ cotwins were remarkably similar in most echocardiographic measurements including epicardial fat (intraclass correlation 0.63, p = 0.0004). However, in the discordant pairs, the obese cotwins (16.5 kg, 23% heavier) had 26% more epicardial fat (p = 0.0029) than nonobese cotwins. They also had significantly larger atrial and left ventricular dimensions. Epicardial fat correlated with VAT (r = 0.49, p = 0.02) in individual twins and when using intrapair differences of measurements within pairs (r = 0.39, p = 0.06). In multiple regression analyses including abdominal subcutaneous tissue, VAT, and liver fat, high-sensitivity C-reactive protein was the only factor that remained significantly associated with epicardial fat in individual twins and within pairs. In conclusion, subjects who share the same genes seem to have similar cardiac dimensions. However, acquired obesity increases epicardial fat independent of genetic factors. The close relation between epicardial fat and low-grade inflammation is likely to contribute to the development of cardiovascular disease in obesity.

Promoter methylation of serotonin transporter gene is associated with obesity measures: a monozygotic twin study

Objective:Epigenetic mechanisms are increasingly being recognized as an important factor for obesity. The serotonin transporter gene (SLC6A4) has a critical role in regulating food intake, body weight and energy balance. This study examines the potential association between SLC6A4 promoter methylation and obesity measures in a monozygotic (MZ) twin sample.Methods:We studied 84 MZ twin pairs drawn from the Vietnam Era Twin Registry. Obesity measures include body mass index (BMI), body weight, waist circumference (WC) and waist-hip ratio (WHR). The SLC6A4 promoter methylation profile in peripheral blood leukocytes was quantified by bisulfite pyrosequencing. The association between methylation variation and obesity parameters was examined by mixed-model regression and matched pair analysis, adjusting for age, smoking, alcohol consumption, physical activity and total daily energy intake. Multiple testing was controlled using the adjusted false discovery rate (q-value).Results:Mean methylation level was positively correlated with BMI (r=0.29; P=0.0002), body weight (r=0.31; P<0.0001) and WC (r=0.20; P=0.009), but not WHR. Intra-pair differences in mean methylation were significantly correlated with intra-pair differences in BMI, body weight and WC, but not WHR. On average, a 1% increase in mean methylation was associated with 0.33 kg m−2 increase in BMI (95% CI: 0.02–0.65; P=0.03), 1.16 kg increase in body weight (95% CI, 0.16–2.16; P=0.02) and 0.78 cm increase in WC (95% CI, 0.05–1.50; P=0.03) after controlling for potential confounders.Conclusions:SLC6A4 promoter hypermethylation is significantly associated with an increased prevalence of obesity within a MZ twin study.

Global DNA methylation is associated with insulin resistance: a monozygotic twin study.

Insulin resistance (IR), the hallmark of type 2 diabetes, may be under epigenetic control. This study examines the association between global DNA methylation and IR using 84 monozygotic twin pairs. IR was estimated using homeostasis model assessment (HOMA). Global DNA methylation of Alu repeats in peripheral blood leukocytes was quantified by bisulfite pyrosequencing. The association between global DNA methylation and IR was examined using generalized estimating equation (GEE) and within–twin pair analyses, adjusting for potential confounders. Results show that methylation levels at all four CpG sites were individually associated with IR by GEE (all false discovery rate–adjusted P values ≤0.026). A 10% increase in mean Alu methylation was associated with an increase of 4.55 units (95% CI 2.38–6.73) in HOMA. Intrapair difference in IR was significantly associated with intrapair difference in global methylation level. A 10% increase in the difference in mean Alu methylation was associated with an increase of 4.54 units (0.34–8.71; P = 0.036) in the difference in HOMA. Confirmation of the results by intrapair analyses suggests that genetic factors do not confound the association between global DNA methylation and IR. Exclusion of twins taking diabetes medication (n = 17) did not change our results.