Genomic imprinting: A missing piece of the Multiple Sclerosis puzzle?

Evidence for parent-of-origin effects in complex diseases such as Multiple Sclerosis (MS) strongly suggests a role for epigenetic mechanisms in their pathogenesis. In this review, we describe the importance of accounting for parent-of-origin when identifying new risk variants for complex diseases and discuss how genomic imprinting, one of the best-characterized epigenetic mechanisms causing parent-of-origin effects, may impact etiology of complex diseases. While the role of imprinted genes in growth and development is well established, the contribution and molecular mechanisms underlying the impact of genomic imprinting in immune functions and inflammatory diseases are still largely unknown. Here we discuss emerging roles of imprinted genes in the regulation of inflammatory responses with a particular focus on the Dlk1 cluster that has been implicated in etiology of experimental MS-like disease and Type 1 Diabetes. Moreover, we speculate on the potential wider impact of imprinting via the action of imprinted microRNAs, which are abundantly present in the Dlk1 locus and predicted to fine-tune important immune functions. Finally, we reflect on how unrelated imprinted genes or imprinted genes together with non-imprinted genes can interact in so-called imprinted gene networks (IGN) and suggest that IGNs could partly explain observed parent-of-origin effects in complex diseases. Unveiling the mechanisms of parent-of-origin effects is therefore likely to teach us not only about the etiology of complex diseases but also about the unknown roles of this fascinating phenomenon underlying uneven genetic contribution from our parents.This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.     

New loci regulating rat myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis

Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease in rats that closely mimics many clinical and histopathological aspects of multiple sclerosis. Non-MHC quantitative trait loci regulating myelin oligodendrocyte glycoprotein-induced EAE have previously been identified in the EAE-permissive strain, DA, on rat chromosomes 4, 10, 15, and 18. To find any additional gene loci in another well-known EAE-permissive strain and thereby to assess any genetic heterogeneity in the regulation of the disease, we have performed a genome-wide linkage analysis in a reciprocal (LEW.1AV1 x PVG.1AV1) male/female F(2) population (n = 185). We examined reciprocal crosses, but no parent-of-origin effect was detected. The parental rat strains share the RT1(av1) MHC haplotype; thus, non-MHC genes control differences in EAE susceptibility. We identified Eae16 on chromosome 8 and Eae17 on chromosome 13, significantly linked to EAE phenotypes. Two loci, on chromosomes 1 and 17, respectively showed suggestive linkage to clinical and histopathological EAE phenotypes. Eae16 and Eae17 differ from those found in previously studied strain combinations, thus demonstrating genetic heterogeneity of EAE. Furthermore, we detected a locus-specific parent-of-origin effect with suggestive linkage in Eae17. Further genetic and functional dissection of these loci may disclose critical disease-regulating molecular mechanisms.     

The Use of Mouse Models to Study Epigenetics

Much of what we know about the role of epigenetics in the determination of phenotype has come from studies of inbred mice. Some unusual expression patterns arising from endogenous and transgenic murine alleles, such as the Agouti coat color alleles, have allowed the study of variegation, variable expressivity, transgenerational epigenetic inheritance, parent-of-origin effects, and position effects. These phenomena have taught us much about gene silencing and the probabilistic nature of epigenetic processes. Based on some of these alleles, large-scale mutagenesis screens have broadened our knowledge of epigenetic control by identifying and characterizing novel genes involved in these processes.     

On the detection of imprinted quantitative trait loci in line crosses: effect of linkage disequilibrium

Imprinted quantitative trait loci (QTL) are commonly reported in studies using line-cross designs, especially in livestock species. It was previously shown that such parent-of-origin effects might result from the nonfixation of QTL alleles in one or both parental lines, rather than from genuine molecular parental imprinting. We herein demonstrate that if linkage disequilibrium exists between marker loci and nonfixed QTL, spurious detection of pseudo-imprinting is increased by an additional 40–80% in scenarios mimicking typical livestock situations. This is due to the fact that imprinting can be tested only in F₂ offspring whose sire and dam have distinct marker genotypes. In the case of linkage disequilibrium between markers and QTL, such parents have a higher chance to have distinct QTL genotypes as well, thus resulting in distinct padumnal and madumnal allele substitution effects, i.e., QTL pseudo-imprinting.     

Assessment of parent-of-origin effects in linkage analysis of quantitative traits

Methods are presented for incorporation of parent-of-origin effects into linkage analysis of quantitative traits. The estimated proportion of marker alleles shared identical by descent is first partitioned into a component derived from the mother and a component derived from the father. These parent-specific estimates of allele sharing are used in variance-components or Haseman-Elston methods of linkage analysis so that the effect of the quantitative-trait locus carried on the maternally derived chromosome is potentially different from the effect of the locus on the paternally derived chromosome. Statistics for linkage between trait and marker loci derived from either or both parents are then calculated, as are statistics for testing whether the effect of the maternally derived locus is equal to that of the paternally derived locus. Analyses of data simulated for 956 siblings from 263 nuclear families who had participated in a linkage study revealed that type I error rates for these statistics were generally similar to nominal values. Power to detect an imprinted locus was substantially increased when analyzed with a model allowing for parent-of-origin effects, compared with analyses that assumed equal effects; for example, for an imprinted locus accounting for 30% of the phenotypic variance, the expected LOD score was 4.5 when parent-of-origin effects were incorporated into the analysis, compared with 3.1 when these effects were ignored. The ability to include parent-of-origin effects within linkage analysis of quantitative traits will facilitate genetic dissection of complex traits.     

No Evidence for Enrichment in Schizophrenia for Common Allelic Associations at Imprinted Loci

Most genetic studies assume that the function of a genetic variant is independent of the parent from which it is inherited, but this is not always true. The best known example of parent-of-origin effects arises with respect to alleles at imprinted loci. In classical imprinting, characteristically, either the maternal or paternal copy is expressed, but not both. Only alleles present in one of the parental copies of the gene, the expressed copy, is likely to contribute to disease. It has been postulated that imprinting is important in central nervous system development, and that consequently, imprinted loci may be involved in schizophrenia. If this is true, allowing for parent-of-origin effects might be important in genetic studies of schizophrenia. Here, we use genome-wide association data from one of the world’s largest samples (N = 695) of parent schizophrenia-offspring trios to test for parent-of-origin effects. To maximise power, we restricted our analyses to test two main hypotheses. If imprinting plays a disproportionate role in schizophrenia susceptibility, we postulated a) that alleles showing robust evidence for association to schizophrenia from previous genome-wide association studies should be enriched for parent-of-origin effects and b) that genes at loci imprinted in humans or mice should be enriched both for genome-wide significant associations, and in our sample, for parent-of-origin effects. Neither prediction was supported in the present study. We have shown, that it is unlikely that parent-of-origin effects or imprinting play particularly important roles in schizophrenia, although our findings do not exclude such effects at specific loci nor do they exclude such effects among rare alleles.     

Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region

Background

The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.

Results

A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Δ5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Δ5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Δ5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.

Conclusion

These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Δ5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.     

MHC susceptibility genes to IgA deficiency are located in different regions on different HLA haplotypes

Familial predisposition to IgA deficiency (IgAD) suggests that genetic factors influence susceptibility. Most studies support a polygenic inheritance with a susceptibility locus (designated IGAD1) in the MHC, but its exact location is still controversial. This study aimed to map the predisposing IGAD1 locus (or loci) within the MHC by investigating the pattern of association of the disease with several markers in the region. DNA-based techniques were used to type individual alleles of four polymorphic HLA genes (HLA-DR, -DQA1, -DQB1, and HLA-B), six microsatellites (all located between HLA-DR and HLA-B), and three single nucleotide polymorphisms on the TNF gene. The frequencies of these alleles were compared among ethnically matched populations comprising 182 patients and 343 controls. Additionally, we investigated parents and siblings of 100 of these patients. All four parental haplotypes were established in each family (n = 400), and transmission disequilibrium tests were performed. Surprisingly, our results did not support the hypothesis of a unique susceptibility gene being shared by all MHC susceptibility haplotypes. On HLA-DR1 and -DR7-positive haplotypes IGAD1 mapped to the class II region, whereas on haplotypes carrying HLA-DR3 the susceptibility locus mapped to the telomeric end of the class III region, as reported previously. Our results show how, in complex diseases, individuals may be affected for different genetic reasons and a single linkage signal to a region of a chromosome may actually be the result of disease-predisposing alleles in different linked genes in different pedigrees.     

The analysis of parental origin of alleles may detect susceptibility loci for complex disorders.

The phenomenon of genomic imprinting describes the differential behavior of genes depending on their parental origin, and has been demonstrated in a few rare genetic disorders. In complex diseases, parent-of-origin effects have not been systematically studied, although there may be heuristic value in such an approach. Data from a genome scan performed using 356 affected sibling pair families with type 1 diabetes were examined looking for evidence of excess sharing of either maternal or paternal alleles. At the insulin gene (IDDM2), evidence for excess sharing of alleles transmitted from mothers was detected, which is consistent with transmission disequilibrium results published elsewhere. We also identified additional loci that demonstrate allele sharing predominantly from one parent: IDDM8 shows a paternal origin effect, IDDM10 shows a maternal effect, and a locus on chromosome 16q demonstrates a paternal effect. We have also evaluated these loci for confounding by differences in sex-specific meiotic recombination by performing linkage analysis using sex-specific genetic maps. The analysis of the parental origin of shared alleles from genome scans of complex disorders may provide additional evidence for linkage for known loci, help identify regions containing additional susceptibility loci, and assist the cloning of the genes involved.     

Epigenetics and Sex-Specific Fitness: An Experimental Test Using Male-Limited Evolution in Drosophila melanogaster

When males and females have different fitness optima for the same trait but share loci, intralocus sexual conflict is likely to occur. Epigenetic mechanisms such as genomic imprinting (in which expression is altered according to parent-of-origin) and sex-specific maternal effects have been suggested as ways by which this conflict can be resolved. However these ideas have not yet been empirically tested. We designed an experimental evolution protocol in Drosophila melanogaster that enabled us to look for epigenetic effects on the X-chromosome–a hotspot for sexually antagonistic loci. We used special compound-X females to enforce father-to-son transmission of the X-chromosome for many generations, and compared fitness and gene expression levels between Control males, males with a Control X-chromosome that had undergone one generation of father-son transmission, and males with an X-chromosome that had undergone many generations of father-son transmission. Fitness differences were dramatic, with experimentally-evolved males approximately 20% greater than controls, and with males inheriting a non-evolved X from their father about 20% lower than controls. These data are consistent with both strong intralocus sexual conflict and misimprinting of the X-chromosome under paternal inheritance. However, expression differences suggested that reduced fitness under paternal X inheritance was largely due to deleterious maternal effects. Our data confirm the sexually-antagonistic nature of Drosophila’s X-chromosome and suggest that the response to male-limited X-chromosome evolution entails compensatory evolution for maternal effects, and perhaps modification of other epigenetic effects via coevolution of the sex chromosomes.     

Evidence for Linkage of Bipolar Disorder to Chromosome 18 with a Parent-of-Origin Effect

A susceptibility gene on chromosome 18 and a parent-of-origin effect have been suggested for bipolar affective disorder (BPAD). We have studied 28 nuclear families selected for apparent unilineal transmission of the BPAD phenotype, by using 31 polymorphic markers spanning chromosome 18. Evidence for linkage was tested with affected-sib-pair and LOD score methods under two definitions of the affected phenotype. The affected-sib-pair analyses indicated excess allele sharing for markers on 18p within the region reported previously. The greatest sharing was at D18S37: 64% in bipolar and recurrent unipolar (RUP) sib pairs (P = .0006). In addition, excess sharing of the paternally, but not maternally, transmitted alleles was observed at three markers on 18q: at D18S41, 51 bipolar and RUP sib pairs were concordant for paternally transmitted alleles, and 21 pairs were discordant (P = .0004). The evidence for linkage to loci on both 18p and 18q was strongest in the 11 paternal pedigrees, i.e., those in which the father or one of the father's sibs is affected. In these pedigrees, the greatest allele sharing (81%; P = .00002) and the highest LOD score (3.51; θ = 0.0) were observed at D18S41. Our results provide further support for linkage of BPAD to chromosome 18 and the first molecular evidence for a parent-of-origin effect operating in this disorder. The number of loci involved, and their precise location, require further study.     

Hérédité et épigénétique: un rôle inattendu pour l’ARN

Although Mendel’s first laws explain the transmission of most characteristics, there has recently been a renewed interest in the notion that DNA is not the sole determinant of our inherited phenotype. Human epidemiology studies and animal and plant genetic studies have provided evidence that epigenetic information (“epigenetic” describes an inherited effect on chromosome or gene function that is not accompanied by any alteration of the nucleotide sequence) can be inherited from parents to offspring. Most of the mechanisms involved in epigenetic “memory” are paramutation events, which are heritable epigenetic changes in the phenotype of a “paramutable” allele. Initially demonstrated in plants, paramutation is defined as an interaction between two alleles of a single locus that results in heritable changes of one allele that is induced by the other. The authors describe an unexpected example of paramutation in the mouse revealed by a recent analysis of an epigenetic variation modulating expression of theKit locus. The progeny of hétérozygote intercrosses (carrying one mutant and one wild-type allele) showed persistence of the white patches (characteristic of hétérozygotes) in the homozygous Kit^+/+ progeny. The DNA sequences of the two wild-type alleles were structurally normal, revealing an epigenetic modification. Further investigations showed that RNA and microRNA, released by sperm, mediate this epigenetic inheritance. The molecular mechanisms involved in this unexpected mode of inheritance and the role of RNA molecules in the spermatozoon head as possible vectors for the hereditary transfer of such modifications — implying that paternal inheritance is not limited to just one haploid copy of the genome — are still a matter of debate. Paramutations may be considered to be one possibility of epigenetic modification in the case of familial disease predispositions not fully explained by Mendelian analysis.     

Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia

Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia.     

Detection of parent-of-origin effects for quantitative traits using general pedigree data

Genomic imprinting is a genetic phenomenon in which certain alleles are differentially expressed in a parent-of-origin-specific manner, and plays an important role in the study of complex traits. For a diallelic marker locus in human, the parental-asymmetry tests Q-PAT(c) with any constant c were developed to detect parent-of-origin effects for quantitative traits. However, these methods can only be applied to deal with nuclear families and thus are not suitable for extended pedigrees. In this study, by making no assumption about the distribution of the quantitative trait, we first propose the pedigree parental-asymmetry tests Q-PPAT(c) with any constant c for quantitative traits to test for parent-of-origin effects based on nuclear families with complete information from general pedigree data, in the presence of association between marker alleles under study and quantitative traits. When there are any genotypes missing in pedigrees, we utilize Monte Carlo (MC) sampling and estimation and develop the Q-MCPPAT(c) statistics to test for parent-of-origin effects. Various simulation studies are conducted to assess the performance of the proposed methods, for different sample sizes, genotype missing rates, degrees of imprinting effects and population models. Simulation results show that the proposed methods control the size well under the null hypothesis of no parent-of-origin effects and Q-PPAT(c) are robust to population stratification. In addition, the power comparison demonstrates that Q-PPAT(c) and Q-MCPPAT(c) for pedigree data are much more powerful than Q-PAT(c) only using two-generation nuclear families selected from extended pedigrees.     

Germline-derived DNA methylation and early embryo epigenetic reprogramming: The selected survival of imprints

DNA methylation is an essential epigenetic mechanism involved in many essential cellular processes. During development epigenetic reprograming takes place during gametogenesis and then again in the pre-implantation embryo. These two reprograming windows ensure genome-wide removal of methylation in the primordial germ cells so that sex-specific signatures can be acquired in the sperm and oocyte. Following fertilization the majority of this epigenetic information is erased to give the developing embryo an epigenetic profile coherent with pluripotency. It is estimated that ∼65% of the genome is differentially methylated between the gametes, however following embryonic reprogramming only parent-of-origin methylation at known imprinted loci remains. This suggests that trans-acting factors such as Zfp57 can discriminate imprinted differentially methylated regions (DMRs) from the thousands of CpG rich regions that are differentially marked in the gametes. Recently transient imprinted DMRs have been identified suggesting that these loci are also protected from pre-implantation reprograming but succumb to de novo remethylation at the implantation stage. This highlights that “ubiquitous” imprinted loci are also resilient to gaining methylation by protecting their unmethylated alleles. In this review I examine the processes involved in epigenetic reprograming and the mechanisms that ensure allelic methylation at imprinted loci is retained throughout the life of the organism, discussing the critical differences between mouse and humans.This article is part of a Directed Issue entitled: Epigenetics Dynamics in development and disease.     

On the allelic spectrum of human disease

Human disease genes show enormous variation in their allelic spectra; that is, in the number and population frequency of the disease-predisposing alleles at the loci. For some genes, there are a few predominant disease alleles. For others, there is a wide range of disease alleles, each relatively rare. The allelic spectrum is important: disease genes with only a few deleterious alleles can be more readily identified and are more amenable to clinical testing. Here, we weave together strands from the human mutation and population genetics literature to provide a framework for understanding and predicting the allelic spectra of disease genes. The theory does a reasonable job for diseases where the genetic etiology is well understood. It also has bearing on the Common Disease/Common Variants (CD/CV) hypothesis, predicting that at loci where the total frequency of disease alleles is not too small, disease loci will have relatively simple spectra.     

Simple inheritance of color and pattern polymorphism in the steppe grasshopper Chorthippus dorsatus

The green–brown polymorphism of grasshoppers and bush-crickets represents one of the most penetrant polymorphisms in any group of organisms. This poses the question of why the polymorphism is shared across species and how it is maintained. There is mixed evidence for whether and in which species it is environmentally or genetically determined in Orthoptera. We report breeding experiments with the steppe grasshopper Chorthippus dorsatus, a polymorphic species for the presence and distribution of green body parts. Morph ratios did not differ between sexes, and we find no evidence that the rearing environment (crowding and habitat complexity) affected the polymorphism. However, we find strong evidence for genetic determination for the presence/absence of green and its distribution. Results are most parsimoniously explained by three autosomal loci with two alleles each and simple dominance effects: one locus influencing the ability to show green color, with a dominant allele for green; a locus with a recessive allele suppressing green on the dorsal side; and a locus with a recessive allele suppressing green on the lateral side. Our results contribute to the emerging contrast between the simple genetic inheritance of green–brown polymorphisms in the subfamily Gomphocerinae and environmental determination in other subfamilies of grasshoppers. In three out of four species of Gomphocerinae studied so far, the results suggest one or a few loci with a dominance of alleles allowing the occurrence of green. This supports the idea that brown individuals differ from green individuals by homozygosity for loss-of-function alleles preventing green pigment production or deposition.Subject terms: Quantitative trait loci, Quantitative trait