Extensive variation and low heritability of DNA methylation identified in a twin study

Disturbance of DNA methylation leading to aberrant gene expression has been implicated in the etiology of many diseases. Whereas variation at the genetic level has been studied extensively, less is known about the extent and function of epigenetic variation. To explore variation and heritability of DNA methylation, we performed bisulfite sequencing of 1760 CpG sites in 186 regions in the human major histocompatibility complex (MHC) in CD4+ lymphocytes from 49 monozygotic (MZ) and 40 dizygotic (DZ) twin pairs. Individuals show extensive variation in DNA methylation both between and within regions. In addition, many regions also have a complex pattern of variation. Globally, there appears to be a bimodal distribution of DNA methylation in the regions, but a significant fraction of the CpG sites are also heterogeneously methylated. Classification of regions into CpG islands (intragenic and intergenic), 5' end of genes not associated with a defined CpG island, conserved noncoding regions, and random CpG sites shows region-type differences in variation and heritability. Analyses revealed slightly lower intra-pair differences among MZ than among DZ pairs, suggesting some genetic influences on DNA methylation variation, with most of the variance attributed to nongenetic factors. Overall, heritability estimates of DNA methylation were low. Our heritability estimates are, however, somewhat deflated due to the presence of batch effects that artificially inflate the estimates of shared environment.     

Hypomethylation within gene promoter regions and type 1 diabetes in discordant monozygotic twins

Genetic susceptibility to type 1 diabetes (T1D) is well supported by epidemiologic evidence; however, disease risk cannot be entirely explained by established genetic variants identified so far. This study addresses the question of whether epigenetic modification of the inherited DNA sequence may contribute to T1D susceptibility. Using the Infinium HumanMethylation450 BeadChip array (450k), a total of seven long-term disease-discordant monozygotic (MZ) twin pairs and five pairs of HLA-identical, disease-discordant non-twin siblings (NTS) were examined for associations between DNA methylation (DNAm) and T1D. Strong evidence for global hypomethylation of CpG sites within promoter regions in MZ twins with TID compared to twins without T1D was observed. DNA methylation data were then grouped into three categories of CpG sites for further analysis, including those within: 1) the major histocompatibility complex (MHC) region, 2) non-MHC genes with reported T1D association through genome wide association studies (GWAS), and 3) the epigenome, or remainder of sites that did not include MHC and T1D associated genes. Initial results showed modest methylation differences between discordant MZ twins for the MHC region and T1D-associated CpG sites, BACH2, INS-IGF2, and CLEC16A (DNAm difference range: 2.2%–5.0%). In the epigenome CpG set, the greatest methylation differences were observed in MAGI2, FANCC, and PCDHB16, (DNAm difference range: 6.9%–16.1%). These findings were not observed in the HLA-identical NTS pairs. Targeted pyrosequencing of five candidate CpG loci identified using the 450k array in the original discordant MZ twins produced similar results using control DNA samples, indicating strong agreement between the two DNA methylation profiling platforms. However, findings for the top five candidate CpG loci were not replicated in six additional T1D-discordant MZ twin pairs. Our results indicate global DNA hypomethylation within gene promoter regions may contribute to T1D; however, findings do not support the involvement of large DNAm differences at single CpG sites alone in T1D.     

The Impact of Variation in Twin Relatedness on Estimates of Heritability and Environmental Influences

By taking advantage of the natural variation in genetic relatedness among identical (monozygotic: MZ) and fraternal (dizygotic: DZ) twins, twin studies are able to estimate genetic and environmental contributions to complex human behaviors. Recently concerns have been raised about the accuracy of twin studies in light of findings of genetic and epigenetic changes in twins. One of the concerns raised is that MZ twins are not 100% genetically and epigenetically similar because they show variations in their genomes and epigenomes leading to inaccurate estimates of heritability. This article presents findings from a simulation study that examined the degree of bias in estimates of heritability and environmentality when the genetic and epigenetic similarity of MZ twins differs from 1.00 and when the genetic and epigenetic similarity of DZ twins differs from 0.50. The findings suggest that in the standard biometric model when MZ or DZ twin similarity differs from 1.00 or 0.50, respectively, the variance that should be attributed to genetic influences is instead attributed to nonshared environmental influences, thus deflating the estimates of genetic influences and inflating the estimates of nonshared environmental influences. Although estimates of genetic and nonshared environmental influences from the standard biometric model were found to deviate from “true” values, the bias was usually smaller than 10% points indicating that the interpretations of findings from previous twin studies are mostly correct.     

X-chromosome inactivation is mostly random in placental tissues of female monozygotic twins and triplets

Patterns of X-chromosome inactivation in chorion, amnion, and cord from 79 pairs of twins were examined. Seven sets of triplets were included in the analysis, both as twin pairs and triplets. Twins were stratified as dizygotic (DZ), monozygotic (MZ), monochorionic, and dichorionic and were selected for birth weight discordance, discordance for congenital anomalies, twin-twin transfusion syndrome, and various patterns of vascular anastomosis. X-inactivation was predominantly symmetric. Chorion was the most likely tissue to show asymmetric X-inactivation and was found most frequently in MZ dichorionic twins. There was no correlation of X-inactivation pattern with the selected clinical criteria. This study does not confirm that asymmetric X-inactivation in embryonic tissues is a common phenomenon in female twins, including monozygotic twins. © 1996 Wiley-Liss, Inc.     

Evidence for monozygotic twin (MZ) discordance in methylation level at two CpG sites in the promoter region of the catechol-O-methyltransferase (COMT) gene.

Monozygotic (MZ) twin concordance for a range of psychiatric conditions is rarely 100%. It has been suggested that epigenetic factors, such as DNA methylation, may account for a proportion of the variation in behavioral traits observed between these genetically identical individuals. In this study we have quantitatively assessed the methylation status of two CpG sites in the promoter region of the COMT gene in 12 MZ twins-pairs discordant for birth weight, but otherwise clinically unaffected. DNA was obtained at age 5-years using buccal swabs, and modified using sodium-bisulfite treatment. Methylation profiles were assessed using Pyrosequencing, a technology enabling the precise degree of methylation to be assessed at any CpG site. We found that the degree of methylation at the two CpG sites was highly correlated, but there was considerable variation in the concordance of methylation levels between MZ twin-pairs. Some MZ twin-pairs showed a high degree of methylation concordance, whereas others differed markedly in their methylation profiles. Such epigenetic variation between genetically identical individuals may play a key role in the etiology of psychopathology, and explain the incomplete phenotypic concordance observed in MZ twins.     

Genetic, environmental and stochastic factors in monozygotic twin discordance with a focus on epigenetic differences

ABSTRACT: Genetic-epidemiological studies on monozygotic (MZ) twins have been used for decades to tease out the relative contributions of genes and the environment to a trait. Phenotypic discordance in MZ twins has traditionally been ascribed to non-shared environmental factors acting after birth, however recent data indicate that this explanation is far too simple. In this paper, we review other reasons for discordance, including differences in the in utero environment, genetic mosaicism, and stochastic factors, focusing particularly on epigenetic discordance. Epigenetic differences are gaining increasing recognition. Although it is clear that in specific cases epigenetic alterations provide a causal factor in disease etiology, the overall significance of epigenetics in twin discordance remains unclear. It is also challenging to determine the causality and relative contributions of environmental, genetic, and stochastic factors to epigenetic variability. Epigenomic profiling studies have recently shed more light on the dynamics of temporal methylation change and methylome heritability, yet have not given a definite answer regarding their relevance to disease, because of limitations in establishing causality. Here, we explore the subject of epigenetics as another component in human phenotypic variability and its links to disease focusing particularly on evidence from MZ twin studies.     

Epigenetics Lessons from Twins: Prospects for Autoimmune Disease

The existence of phenotypic differences between monozygotic (MZ) twins is a prime case where the relationship between genetic determinants and environmental factors is illustrated. Although virtually identical from a genetic point of view, MZ twins show a variable degree of discordance with respect to different features including susceptibility to disease. Discordance has frequently been interpreted in terms of the impact of the environment with genetics. In this sense, accumulated evidence supports the notion that environmental factors can have a long-term effect on epigenetic profiles and influence the susceptibility to disease. In relation with autoimmune diseases, the identification of DNA methylation changes in individuals who develop the disease, and the influence of inhibitors of DNA methyltransferases and histone modification enzymes in the development of autoimmunity are attracting the attention of researchers in the epigenetics field. In this context, the study of discordant MZ twins constitutes an attractive model to further investigate the epigenetic mechanisms involved in their development as well as to dissect the contribution of environmental traits. The implications of novel strategies to map epigenetic profiles and how the use of MZ twins can contribute to dissect the epigenetic component of autoimmune disease are discussed.     

Laterality in twins: The relationship between handedness and hemispheric asymmetry for speech

The present study examined the relationship between dichotic listening performance and handedness in twins. The 53 monozygotic (MZ) and 35 dizygotic (DZ) pairs concordant for right-handedness displayed ear asymmetries and total correct scores comparable to those found in right-handed singletons. The left- and right-handed members of the 19 MZ and 8 DZ pairs discordant for handedness were also similar to left- and right-handed singletons, respectively, with regard to ear asymmetry and overall performance. These data demonstrate that the relationship between handedness and brain organization observed in both MZ and DZ twins is similar to that found in the singleton population. The pattern of intraclass correlations obtained suggests that handedness discordance may be associated with greater differences in ear asymmetry within MZ pairs than within DZ pairs, possibly reflecting differences in the etiology of discordance for handedness in the two groups. In contrast, MZ cotwins were more similar than DZ cotwins, regardless of handedness, when total correct performance was measured.This research was supported in part by NIH Grant RO3MH2710101.     

Differentially Methylated Genomic Regions in Birth‐Weight Discordant Twin Pairs

Poor nutrition during critical growth phases may alter the structural and physiologic development of vital organs thus “programming” the susceptibility to adult‐onset diseases and disease‐related health conditions. Epigenome‐wide association studies have been performed in birth‐weight discordant twin pairs to find evidence for such “programming” effects, but no significant results emerged. We further investigated this issue using a new computational approach: Instead of probing single genomic sites for significant alterations in epigenetic marks, we scan for differentially methylated genomic regions. Whole genome DNA methylation levels were measured in whole blood from 150 pairs of adult identical twins discordant for birth‐weight. Intrapair differential DNA methylation was associated with qualitative (large or small) and quantitative (percentage) birth‐weight discordance at each genomic site using regression models adjusting for age and sex. Based on the regression results, genomic regions with consistent alteration patterns of DNA methylation were located and tested for significant robustness using computational permutation tests. This yielded an interesting genomic region on chromosome 1, which is significantly differentially methylated for quantitative birth‐weight discordance. The region covers two genes (TYW3 and CRYZ) both reportedly associated with metabolism. We conclude that prenatal conditions for birth‐weight discordance may result in persistent epigenetic modifications potentially affecting even adult health.     

X-chromosome inactivation patterns in monozygotic twins and sib pairs discordant for nonsyndromic cleft lip and/or palate

Nonsyndromic clefts of the lip and/or palate are common birth defects with a strong genetic component. Based on unequal gender ratios for clefting phenotypes, evidence for linkage to the X chromosome and the occurrence of several X-linked clefting syndromes, we investigated the role of skewed X chromosome inactivation (XCI) in orofacial clefts. Our samples consisted of female monozygotic (MZ) twins (n = 8) and sister pairs (n = 152) discordant for nonsyndromic clefting. We measured the XCI pattern in peripheral blood lymphocyte DNA using a methylation based androgen receptor gene assay. Skewing of XCI was defined as the deviation in inactivation pattern from a 50:50 ratio. Our analysis revealed no significant difference in the degree of skewing between twin pairs (P = 0.3). However, borderline significant differences were observed in the sister pairs (P = 0.02), with the cleft lip with cleft palate group showing the most significant result (P = 0.01). We did not find evidence for involvement of skewed XCI in the discordance for clefting in our sample of female MZ twins. However, results from the paired sister study suggest the potential contribution of skewed XCI to orofacial clefting, particularly cleft lip and palate.     

Genetic and environmental influences on birthweight in a sample of Korean twins

This study is the first report of genetic and environmental influences on birthweight using Korean twins. The sample consisted of 255 monozygotic (MZ) and 178 dizygotic (DZ) twin pairs drawn from the Seoul Twin Family Study. Intraclass twin correlations were computed for the twins' birthweights obtained from parents (typically mothers) of the twins. To estimate genetic and shared and nonshared environmental influences on birthweight, standard univariate model-fitting analyses were performed using a software, Mx. For each gender, MZ twin correlations were higher than DZ twin correlations, suggesting existence of genetic influences on birthweight; however, DZ twin correlations were higher than half the MZ twin correlations, indicating that shared environmental factors are also important. For each zygosity, twin correlations were not significantly different between males and females, implicating that genes and environments that cause individual differences in birthweight may not vary between males and females. Model-fitting analyses based on the data pooled across gender yielded estimates of 17% for genetic, 60% for shared environmental, and 23% for nonshared environmental influences on birthweight.     

Hypermethylation of CpG islands is more prevalent than hypomethylation across the entire genome in breast carcinogenesis

This study aimed to establish a high-throughput, genome-wide and non-gene-specific approach to assess the methylation status of multiple CpG islands in parallel and employ it to detect the CpG island methylation profiling alterations in breast carcinogenesis. We used methylation-sensitive restriction fingerprint (MSRF) to screen the permutations of primers that could detect varied and specific methylation profiling in genomic DNA isolated from four different cell lines. Five permutations of nine arbitrary primers were determined for the following experiments based on the above test. We then examined the methylation profiling alterations of CpG islands in 31 breast cancer tissue samples relative to their adjacent non-neoplastic tissues with modified MSRF that replaced silver staining with denatured high-performance liquid chromatography for size fraction. We found that two pairs of primers could reveal specific alterations of CpG methylation in the examined tissues, and 83.9% (26/31) of breast cancer tissues exhibited specific CpG island methylation profiling relative to their adjacent non-neoplastic tissues. Size fraction analysis revealed that hypermethylation of CpG islands was responsible for the aberrant methylation profiling in breast cancer tissues. Our work not only established a relative high-throughput, genome-wide and economic method to detect methylation alterations of CpG island profiling, but also revealed that hypermethylation of CpG islands was more prevalent than hypomethylation across the entire genome in our examined cancer tissues. The methylation profiling alterations revealed by two primer pairs used in the present study might be a novel marker for breast cancer.     

Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder

Studies of the major psychoses, schizophrenia (SZ) and bipolar disorder (BD), have traditionally focused on genetic and environmental risk factors, although more recent work has highlighted an additional role for epigenetic processes in mediating susceptibility. Since monozygotic (MZ) twins share a common DNA sequence, their study represents an ideal design for investigating the contribution of epigenetic factors to disease etiology. We performed a genome-wide analysis of DNA methylation on peripheral blood DNA samples obtained from a unique sample of MZ twin pairs discordant for major psychosis. Numerous loci demonstrated disease-associated DNA methylation differences between twins discordant for SZ and BD individually, and together as a combined major psychosis group. Pathway analysis of our top loci highlighted a significant enrichment of epigenetic changes in biological networks and pathways directly relevant to psychiatric disorder and neurodevelopment. The top psychosis-associated, differentially methylated region, significantly hypomethylated in affected twins, was located in the promoter of ST6GALNAC1 overlapping a previously reported rare genomic duplication observed in SZ. The mean DNA methylation difference at this locus was 6%, but there was considerable heterogeneity between families, with some twin pairs showing a 20% difference in methylation. We subsequently assessed this region in an independent sample of postmortem brain tissue from affected individuals and controls, finding marked hypomethylation (>25%) in a subset of psychosis patients. Overall, our data provide further evidence to support a role for DNA methylation differences in mediating phenotypic differences between MZ twins and in the etiology of both SZ and BD.     

Epigenetic contributors to the discordance of monozygotic twins

Human monozygotic (MZ) twins estimated to occur once in 250 live births, result from an errant decision by embryonic cell(s) to develop as separate embryos. They are considered genetically identical and any phenotypic discordance between them has been used to implicate the role of environment. More recent literature, however, has questioned these assumptions but the frequency and the nature of any genetic discordance between MZ twins remains poorly understood. We will review published cases of phenotypic and genetic discordance between monozygotic twins to argue that not all discordance between such twins is due to differences in environment. The causes of reduced concordance between MZ twins remains poorly understood. They represent among the challenging aspects of the genetics of complex multi-factorial traits and diseases. A number of questions regarding the published results on MZ twins merit a re-assessment in the light of modern molecular insight of the human genome. Such an assessment is needed in directing future studies on MZ twins. In particular, we will deal with the origin, development, genetic and epigenetic factors that may have implications in discordance of the MZ twin pairs.     

Intra-pair similarity in physical growth of monozygotic and of dizygotic twins during puberty

The growth of 323 pairs of twins (94 MZ pairs, 133 DZ like-sexed pairs and 97 DZ unlike-sexed pairs) born in 1954/55 was studied longitudinally from 10 to 18 years for boys and 10 to 16 years for girls. Within-pair comparisons for height and weight have been made for MZ twins and DZ like-sexed twins. A significantly higher concordance in height was found for MZ pairs than for DZ pairs during puberty, for both boys and girls. Yearly height increments were also more similar for the MZ pairs, showing that the height spurt occurs more simultaneously for MZ twins in comparison to DZ twins. Height seems to be a strongly genetically regulated variable. The MZ twins also show a high concordance in weight during the whole investigation period. The DZ pairs, however, became progressively less similar in weight. This is most pronounced for the DZ girls, the intra-class correlation for weight decreasing from 0.66 at 10 years to 0.23 at 16 years. This could imply stronger interactional effects for girls than for boys with regard to weight during puberty.     

Intra-pair similarity in physical growth of monozygotic and of dizygotic twins during puberty

The growth of 323 pairs of twins (94 MZ pairs, 133 DZ like-sexed pairs and 97 DZ unlike-sexed pairs) born in 1954/55 was studied longitudinally from 10 to 18 years for boys and 10 to 16 years for girls. Within-pair comparisons for height and weight have been made for MZ twins and DZ like-sexed twins. A significantly higher concordance in height was found for MZ pairs than for DZ pairs during puberty, for both boys and girls. Yearly height increments were also more similar for the MZ pairs, showing that the height spurt occurs more simultaneously for MZ twins in comparison to DZ twins. Height seems to be a strongly genetically regulated variable.

The MZ twins also show a high concordance in weight during the whole investigation period. The DZ pairs, however, became progressively less similar in weight. This is most pronounced for the DZ girls, the intra-class correlation for weight decreasing from 0·66 at 10 years to 0·23 at 16 years. This could imply stronger interactional effects for girls than for boys with regard to weight during puberty.     

Chromosome findings in twins with early-onset autistic disorder

In a twin study of autistic disorder, chromosome analyses were carried out in nine pairs of monozygotic (MZ) twins, two pairs of dizygotic (DZ) twins, one set of MZ triplets, one single twin from a MZ pair, and seven single twins from DZ pairs. All but one of the MZ sets were concordant for autistic disorder; all DZ pairs were discordant. Fragile X(q)(27.3) was found in one pair of MZ twins and in MZ triplets, i.e., in 9% of the population with autistic disorder. A marker chromosome of unknown origin was detected in a male twin with autistic disorder from a discordant DZ pair.     

Genetic analysis of growth curve parameters of body weight,height and head circumference

A sample of 681 Israeli boys and girls, including 355 regular siblings (SB), 112 pairs of dizygotic (DZ) and 51 pairs of monozygotic (MZ) twins, was measured for body weight (WT), length (HT) and head circumference (HC) at birth and during the first year of life. The Count model with three parameters was chosen as the best fitting and most parsimonious function to approximate growth of the studied traits. The curves' fitting parameters were estimated for WT, HT and HC for each individual. To test the assumption that there is a genetic source influencing the pattern of growth for each trait, familial correlations between parameter estimates were computed for MZ, DZ twins and SB. In all instances MZ twins showed the highest within-pair correlation in parameters of growth (from 0.58 to 0.86), while SB showed the lowest ones (from 0.10 to 0.70). Variance decomposition analysis was used to simultaneously assess the contribution of gender, gestational age, additive genetic factor, common sibs and common intrauterine environmental effects on total variance of each studied trait separately. All these sources of variation were statistically significant, though the effect of intrauterine environment played a substantial role in early stages of child physical development, explaining from 18.1% to 70.6% of the total variance of the growth curve parameters. Further analyses are needed to clarify how this environment affects child growth and for how long.     

Birth weight and age at menopause in Australian female twin pairs: exploration of the fetal origin hypothesis

In a twin sample where duration of gestation can be controlled, a specific example of the fetal origins hypothesis concerning association between low birth weight and early age at menopause is explored. The hypothesis is based on the physiologically plausible path from intrauterine growth retardation and reduced numbers of primary follicles to an earlier menopause. The sample comprised 323 Australian female twin pairs where both co-twins had reached menopause naturally and reported on their weight at birth. Regression analysis showed no linear association between the two variables (P = 0.371, r(2) = 0.0009). Intra-pair differences in age at menopause were investigated in the context of relative birth weight of co-twins. In 265 pairs an intra-pair birth weight difference was reported. In monozygotic (MZ) pairs (n = 168) this allowed for control of genetic effects as well as gestation duration. No significant differences dependent on birth weight relative to co-twin were found for age at natural menopause in either MZ or dizygotic (DZ) twin pairs, even in pairs whose birth weights differed markedly. There was some indication that twins with premature ovarian failure were heavier at birth than twins with normal or later menopausal age. We conclude that the hypothesis that lower birth weight is associated with earlier menopause is not supported by our data.