Novel Epigenetic Changes Unveiled by Monozygotic Twins Discordant for Smoking Habits

Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.     

DNA methylation and gene expression changes in monozygotic twins discordant for psoriasis: identification of epigenetically dysregulated genes

Monozygotic (MZ) twins do not show complete concordance for many complex diseases; for example, discordance rates for autoimmune diseases are 20%-80%. MZ discordance indicates a role for epigenetic or environmental factors in disease. We used MZ twins discordant for psoriasis to search for genome-wide differences in DNA methylation and gene expression in CD4(+) and CD8(+) cells using Illumina's HumanMethylation27 and HT-12 expression assays, respectively. Analysis of these data revealed no differentially methylated or expressed genes between co-twins when analyzed separately, although we observed a substantial amount of small differences. However, combined analysis of DNA methylation and gene expression identified genes where differences in DNA methylation between unaffected and affected twins were correlated with differences in gene expression. Several of the top-ranked genes according to significance of the correlation in CD4(+) cells are known to be associated with psoriasis. Further, gene ontology (GO) analysis revealed enrichment of biological processes associated with the immune response and clustering of genes in a biological pathway comprising cytokines and chemokines. These data suggest that DNA methylation is involved in an epigenetic dysregulation of biological pathways involved in the pathogenesis of psoriasis. This is the first study based on data from MZ twins discordant for psoriasis to detect epigenetic alterations that potentially contribute to development of the disease.     

Effects of acquired obesity on endothelial function in monozygotic twins

Objective: To determine whether acquired obesity or accompanying metabolic changes such as adiponectin deficiency, insulin resistance, dyslipidemia, or visceral fat are associated, independent of genetic influences, with endothelial dysfunction by studying young adult monozygotic (MZ) twin pairs discordant for obesity. Research Methods and Procedures: Nine obesity‐discordant (intra‐pair difference in BMI, 3.8 to 10.1 kg/m²) and nine concordant (0 to 2.3 kg/m²) 24‐ to 27‐year‐old MZ twin pairs were identified from a population‐based FinnTwin16‐sample. Endothelial function was measured by blood flow responses to intrabrachial infusions of an endothelium‐dependent (acetylcholine) and an endothelium‐independent (sodium nitroprusside) vasodilator. Whole body insulin sensitivity was measured using the euglycemic insulin clamp technique, and forearm and body composition were measured with magnetic resonance imaging and DXA. In addition, serum levels of adiponectin, high‐sensitivity C‐reactive protein, and lipids were determined. Results: The heavier co‐twins of the discordant pairs had significantly lower whole body insulin sensitivity than the leaner co‐twins. Blood flows/muscle volume during infusions of acetylcholine and sodium nitroprusside were not altered by obesity. However, intra‐pair differences in serum adiponectin, intra‐abdominal fat, and C‐reactive protein were significantly correlated with those in endothelial function. Only the relationship between intra‐pair differences in adiponectin and endothelial function persisted in multiple linear regression analysis. Obesity‐concordant co‐twins had comparable insulin sensitivity and endothelial function. Discussion: In young adult MZ twins discordant for obesity, acquired adiponectin deficiency rather than obesity per se is an independent correlate of endothelial dysfunction.     

Increased DNA methylation at the AXIN1 gene in a monozygotic twin from a pair discordant for a caudal duplication anomaly

The AXIN1 gene has been implicated in caudal duplication anomalies. Its coding region was sequenced in both members of a monozygotic (MZ) twin pair discordant for a caudal duplication anomaly, but no mutation was found. Using bisulfite sequencing, we examined methylation at the promoter region of the AXIN1 gene in these twins and in twin and age-matched singleton controls. Methylation of the promoter region in peripheral blood mononucleated cells was variable among individuals, including MZ pairs. In the MZ pair discordant for the caudal duplication, this region of the affected twin was significantly more methylated than that of the unaffected twin (P < .0001), which was significantly more methylated than those of the controls (P = .02). We have confirmed that this CpG island does function as a promoter in vitro and that its activity is inversely proportional to the extent of methylation. This finding raises the possibility that hypermethylation of the AXIN1 promoter, by mechanisms as yet undetermined, is associated with the malformation. This case may be paradigmatic for some cases of MZ discordance.     

DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.     

Differentiating between monozygotic twins through DNA methylation-specific high-resolution melt curve analysis

Although short tandem repeat profiling is extremely powerful in identifying individuals from crime scene stains, it is unable to differentiate between monozygotic (MZ) twins. Efforts to address this include mutation analysis through whole genome sequencing and through DNA methylation studies. Methylation of DNA is affected by environmental factors; thus, as MZ twins age, their DNA methylation patterns change. This can be characterized by bisulfite treatment followed by pyrosequencing. However, this can be time-consuming and expensive; thus, it is unlikely to be widely used by investigators. If the sequences are different, then in theory the melting temperature should be different. Thus, the aim of this study was to assess whether high-resolution melt curve analysis can be used to differentiate between MZ twins. Five sets of MZ twins provided buccal swabs that underwent extraction, quantification, bisulfite treatment, polymerase chain reaction amplification and high-resolution melting curve analysis targeting two markers, Alu-E2F3 and Alu-SP. Significant differences were observed between all MZ twins targeting Alu-E2F3 and in four of five MZ twins targeting Alu-SP (P < 0.05). Thus, it has been demonstrated that bisulfite treatment followed by high-resolution melting curve analysis could be used to differentiate between MZ twins.     

Potential bias regarding birth weight in historical and contemporary twin data bases.

In this study we examine the hypothesis that monozygotic (MZ) twins in historical databases are less discordant for birth weight due to negative selection of severely discordant MZ twins. Furthermore, we test the hypothesis that MZ twins are less discordant for birth weight when comparing a volunteer based twin registry with a population based twin registry, due to selective registration. Data were available on 3927 twin pairs from the volunteer Australian Twin Registry born before 1964, 3059 volunteer twin pairs from the Netherlands Twin Register born 1987-1989 and 454 Belgian twin pairs from The East Flanders Prospective Twin Survey born 1987-1989. Intrapair relative birth weight differences (RBWD) were computed for MZ and dizygotic (DZ) twins from each twin registry. Comparing birth weight differences between MZ and DZ twins provides support for the hypothesis that MZ twins are subject to a negative selection in historical databases. Furthermore, Australian MZ twins have a lower RBWD compared to Dutch MZ twins when corrected for the RBWD of Australian and Dutch DZ twins, indicating circumstances which only affect MZ twins. Our hypothesis that MZ twins are less discordant for birth weight in a volunteer based twin registry compared to a population based twin registry had to be rejected. We suggest that investigators using historical databases to test the fetal origins hypothesis should be aware of this increased likelihood of selective exclusion of individuals with extreme morphometric parameters at time of birth.     

Acquired liver fat is a key determinant of serum lipid alterations in healthy monozygotic twins

Objective: The effects of acquired obesity on lipid profile and lipoprotein composition in rare BMI‐discordant monozygotic (MZ) twin pairs were studied. Design and Methods: Abdominal fat distribution, liver fat (magnetic resonance imaging and spectroscopy), fasting serum lipid profile (ultracentrifugation, gradient gel‐electrophoresis, and colorimetric enzymatic methods), and lifestyle factors (questionnaires and diaries) were assessed in 15 BMI‐discordant (within‐pair difference [Δ] in BMI >3 kg/m²) and nin concordant (ΔBMI <3 kg/m²) MZ twin pairs, identified from two nationwide cohorts of Finnish twins. Results: Despite a strong similarity of MZ twins in lipid parameters (intra‐class correlations 0.42‐0.90, P < 0.05), concentrations of apolipoprotein B (ApoB), intermediate‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein 3a% (HDL3a%), and HDL3c% were higher (P < 0.05) and those of HDL cholesterol, HDL2‐C, and HDL2b% were lower (P < 0.01) in the heavier co‐twins of BMI‐discordant pairs. The composition of lipoprotein particles was similar in the co‐twins. When BMI‐discordant pairs were further divided into liver fat‐discordant and concordant (based on median for Δliver fat, 2.6%), the adverse lipid profile was only seen in those heavy co‐twins who also had high liver fat. Conversely, BMI‐discordant pairs concordant for liver fat did not differ significantly in lipid parameters. In multivariate analyses controlling for Δsubcutaneous, Δintra‐abdominal fat, sex, Δsmoking and Δphysical activity, Δliver fat was the only independent variable explaining the variation in ΔApoB, Δtotal cholesterol, and ΔLDL‐C concentration. Conclusions: Several pro‐atherogenic changes in the amounts of lipids but not in the composition of lipoprotein particles were observed in acquired obesity. In particular, accumulation of liver fat was associated with lipid disturbances, independent of genetic effects.     

同卵双胞胎在复杂性状DNA甲基化调控机制研究中的应用

同卵双胞胎来源于同一个受精卵,DNA序列基本一致,但在某些重要表型上如复杂疾病,并不完全一样。利用表型不一致的同卵双胞胎进行研究,能在遗传背景、母体效应、年龄性别效应等一致的基础上,深入研究分析复杂性状的表观调控机制。而DNA甲基化是最为稳定的一类表观遗传修饰。在人类中,利用同卵双胞胎对印记异常疾病、精神类疾病、自身免疫病及癌症等疾病的DNA甲基化调控研究已经揭示了多个致病基因,为研究疾病的表观调控以及表观遗传学药物的应用打下了基础。本文着重对同卵双胞胎DNA甲基化状态、DNA甲基化遗传力计算以及复杂性状DNA甲基化调控的研究应用及其进展展开综述,以期为复杂性状表观调控机制研究提供借鉴和参考。     

Genome-Wide DNA Methylation and Gene Expression Analyses of Monozygotic Twins Discordant for Intelligence Levels

Human intelligence, as measured by intelligence quotient (IQ) tests, demonstrates one of the highest heritabilities among human quantitative traits. Nevertheless, studies to identify quantitative trait loci responsible for intelligence face challenges because of the small effect sizes of individual genes. Phenotypically discordant monozygotic (MZ) twins provide a feasible way to minimize the effects of irrelevant genetic and environmental factors, and should yield more interpretable results by finding epigenetic or gene expression differences between twins. Here we conducted array-based genome-wide DNA methylation and gene expression analyses using 17 pairs of healthy MZ twins discordant intelligently. ARHGAP18, related to Rho GTPase, was identified in pair-wise methylation status analysis and validated via direct bisulfite sequencing and quantitative RT-PCR. To perform expression profile analysis, gene set enrichment analysis (GSEA) between the groups of twins with higher IQ and their co-twins revealed up-regulated expression of several ribosome-related genes and DNA replication-related genes in the group with higher IQ. To focus more on individual pairs, we conducted pair-wise GSEA and leading edge analysis, which indicated up-regulated expression of several ion channel-related genes in twins with lower IQ. Our findings implied that these groups of genes may be related to IQ and should shed light on the mechanism underlying human intelligence.     

Utilizing Twins as Controls for Non-Twin Case-Materials in Genome Wide Association Studies

Twin registries around the globe have collected DNA samples from large numbers of monozygotic and dizygotic twins. The twin sample collections are frequently used as controls in disease-specific studies together with non-twins. This approach is unbiased under the hypothesis that twins and singletons are comparable in terms of allele frequencies; i.e. there are no genetic variants associated with being a twin per se. To test this hypothesis we performed a genome-wide association study comparing the allele frequency of 572,352 single nucleotide polymorphisms (SNPs) in 1,413 monozygotic (MZ) and 5,451 dizygotic (DZ) twins with 3,720 healthy singletons. Twins and singletons have been genotyped using the same platform. SNPs showing association with being a twin at P-value < 1 × 10^-5 were selected for replication analysis in 1,492 twins (463 MZ and 1,029 DZ) and 1,880 singletons from Finland. No SNPs reached genome-wide significance (P-value < 5 × 10^-8) in the main analysis combining MZ and DZ twins. In a secondary analysis including only DZ twins two SNPs (rs2033541 close to ADAMTSL1 and rs4149283 close to ABCA1) were genome-wide significant after meta-analysis with the Finnish population. The estimated proportion of variance on the liability scale explained by all SNPs was 0.08 (P-value=0.003) when MZ and DZ were considered together and smaller for MZ (0.06, P-value=0.10) compared to DZ (0.09, P-value=0.003) when analyzed separately. In conclusion, twins and singletons can be used in genetic studies together with general population samples without introducing large bias. Further research is needed to explore genetic variances associated with DZ twinning.     

X chromosome inactivation patterns correlate with fetal-placental anatomy in monozygotic twin pairs: implications for immune relatedness and concordance for autoimmunity.

BACKGROUND: Monozygotic (MZ) twinning is a poorly understood phenomenon that may result in subtle biologic differences between twins, despite their identical inheritance. These differences may in part account for discordant expression of disease in MZ twin pairs. Due to their stochastic nature, differences in X chromosome inactivation patterns are one source of such variation in female MZ twins. MATERIALS AND METHODS: We investigated X chromosome inactivation patterns in the blood of 41 MZ twin pairs based on methylation of the androgen receptor gene using a Hpa II-PCR assay. Twenty-six female MZ twin pairs with autoimmune disease (rheumatoid arthritis or multiple sclerosis) were studied. In addition, we studied 15 newborn female MZ twin pairs who were characterized at birth with respect to the anatomy of chorionic membranes (dichorionic versus monochorionic). RESULTS: We found a strong correlation between dichorionic fetal anatomy and differences in X chromosome inactivation patterns between members of an MZ twin pair. In contrast, all monochorionic twin pairs had closely correlated patterns of X chromosome inactivation. X chromosome inactivation patterns did not distinguish between MZ twin pairs who were concordant or discordant for autoimmune disease. CONCLUSIONS: The highly similar patterns of X chromosome inactivation among monochorionic twin pairs may result from their shared placental blood supply during intrauterine life. Alternatively, these patterns may indicate that X chromosome inactivation occurs before the twinning event in this anatomic subgroup of MZ twins. The data further suggest that these factors do not make a major contribution to the high discordance rates for autoimmune disease in MZ twin pairs.     

Environmental bias in twin studies

Abstract

This study examines the responses of mothers of twin girls about similarities and differences of their monozygotic (MZ) and dizygotic (DZ) twins. After these measures had been completed and scored, the investigator obtained zygosity diagnoses of the twins made by extensive blood‐group analyses. Of the 61 pairs of twins, 11 were misclassified by their mothers. Despite these mothers’ erroneous beliefs about the zygosity of their twins, they described the twins as having similarities and differences appropriate to their true degree of genetic relatedness.     

The Host Genotype and Environment Affect Strain Types of Bifidobacterium longum subsp. longum Inhabiting the Intestinal Tracts of Twins

To investigate the influences of host genotype and environment on Bifidobacterium longum subsp. longum inhabiting human intestines at the strain level, six pairs of twins, divided into two groups (children and adults), were recruited. Each group consisted of two monozygotic (MZ) twin pairs and one dizygotic (DZ) twin pair. Child twins had been living together from birth, while adult twins had been living separately for 5 to 10 years. A total of 345 B. longum subsp. longum isolates obtained from 60 fecal samples from these twins were analyzed by multilocus sequence typing (MLST), and 35 sequence types (STs) were finally acquired. Comparison of strains within and between the twin pairs showed that no strains with identical STs were observed between unrelated individuals or within adult DZ twin pairs. Eight STs were found to be monophyletic, existing within MZ twins and child DZ twins. The similarity of strain types within child cotwins was significantly higher than that within adult cotwins, which indicated that environment was one of the important determinants in B. longum subsp. longum strain types inhabiting human intestines. However, although these differences between MZ and DZ twins were observed, it is still difficult to reach an exact conclusion about the impact of host genotype. This is mainly because of the limited number of subjects tested in the present study and the lack of strain types tracing in the same twin pairs from birth until adulthood.     

Additive,Epistatic, and Environmental Effects Through the Lens of Expression Variability QTL in a Twin Cohort

The expression of a gene can vary across individuals in the general population, as well as between monozygotic twins. This variable expression is assumed to be due to the influence of both genetic and nongenetic factors. Yet little evidence supporting this assumption has been obtained from empirical data. In this study, we used expression data from a large twin cohort to investigate the influences of genetic and nongenetic factors on variable gene expression. We focused on a set of expression variability QTL (evQTL)—i.e., genetic loci associated with the variance, as opposed to the mean, of gene expression. We identified evQTL for 99, 56, and 79 genes in lymphoblastoid cell lines, skin, and fat, respectively. The differences in gene expression, measured by the relative mean difference (RMD), tended to be larger between pairs of dizygotic (DZ) twins than between pairs of monozygotic (MZ) twins, showing that genetic background influenced the expression variability. Furthermore, a more profound RMD was observed between pairs of MZ twins whose genotypes were associated with greater expression variability than the RMD found between pairs of MZ twins whose genotypes were associated with smaller expression variability. This suggests that nongenetic (e.g., environmental) factors contribute to the variable expression. Lastly, we demonstrated that the formation of evQTL is likely due to partial linkages between eQTL SNPs that are additively associated with the mean of gene expression; in most cases, no epistatic effect is involved. Our findings have implications for understanding divergent sources of gene expression variability.