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Several lines of evidence suggest that the presence of the Y chromosome influences DNA methylation of autosomal loci. To better understand the impact of the Y chromosome on autosomal DNA methylation patterns and its contribution to sex bias in methylation, we identified Y chromosome dependent differentially methylated regions (yDMRs) using whole-genome bisulfite sequencing methylation data from livers of mice with different combinations of sex-chromosome complement and gonadal sex. Nearly 90% of the autosomal yDMRs mapped to transposable elements (TEs) and most of them had lower methylation in XY compared to XX or XO mice. Follow-up analyses of four reporter autosomal yDMRs showed that Y-dependent methylation levels were consistent across most somatic tissues but varied in strains with different origins of the Y chromosome, suggesting that genetic variation in the Y chromosome influenced methylation levels of autosomal regions. Mice lacking the q-arm of the Y chromosome (B6.NPYq-2) as well as mice with a loss-of-function mutation in Kdm5d showed no differences in methylation levels compared to wild type mice. In conclusion, the Y-linked modifier of TE methylation is likely to reside on the short arm of Y chromosome and further studies are required to identify this gene.

Sexual dimorphism in gene regulation, including DNA methylation, is the main driver of sexual dimorphism in phenotypes. However, the questions of how and when sex shapes DNA methylation remain unresolved. Recently, using mice with different combinations of genetic and phenotypic sex, we identified sex-associated differentially methylated regions (sDMRs) that depended on the sex phenotype. Focusing on a panel of validated sex-phenotype dependent male- and female-biased sDMRs, we tested the developmental dynamics of sex bias in liver methylation and the impacts of mutations in the androgen receptor, estrogen receptor alpha, or the transcriptional repressor Bcl6 gene. True hermaphrodites that carry both unilateral ovaries and contralateral testes were also tested. Our data show that sex bias in methylation either coincides with or follows sex bias in the expression of sDMR-proximal genes, suggesting that sex bias in gene expression may be required for demethylation at certain sDMRs. Global ablation of AR, ESR1, or a liver-specific loss of BCL6, all alter sDMR methylation, whereas presence of both an ovary and a testis delays the establishment of male-type methylation levels in hermaphrodites. Moreover, the Bcl6-LKO shows dissociation between expression and methylation, suggesting a distinct role of BCL6 in demethylation of intragenic sDMRs.

The present case report describes an Italian family with three affected probands, who exhibited serious mental disability, which has not been associated with other anomalies, except with slight facial dysmorphism. Molecular multigenic analysis for intellectual disability identified a previously unreported variant, p.Ile1765Met (c.5295C>G) in the SNF domain of the ATRX protein (in exon 24). The identified mutation was found in a hemizygous state in all three affected probands and in a heterozygous state in the asymptomatic mother and the female sibling. With respect to the phenotypic similarities found in the patients with those described in previous studies, the consistency in the mode of inheritance and segregation of the mutation, the variant reported in the present case report may be considered as 'likely pathogenic'. To investigate the hypothesis that the preferential transmission of the ATRX mutation observed in this family reflected a general trend, a meta‑analysis into the segregation of ATRX mutations from published pedigrees, following allelic transmission from mothers who are heterozygous carriers to their offspring, was performed. A preferential transmission of the mutant allele to male offspring (58% of males inherited the mutant allele) was found; however, the bias was not statistically significant (P=0.29; χ2 test).

Zona pellucida binding protein 2 (Zpbp2) and ORMDL sphingolipid biosynthesis regulator 3 (Ormdl3), mapped downstream of Zpbp2, were identified as two genes associated with airway hyper-responsiveness (AHR). Ormdl3 gene product has been shown to regulate the biosynthesis of ceramides. Allergic asthma was shown to be associated with an imbalance between very-long-chain ceramides (VLCCs) and long-chain ceramides (LCCs). We hypothesized that Fenretinide can prevent the allergic asthma-induced augmentation of Ormdl3 gene expression, normalize aberrant levels of VLCCs and LCCs, and treat allergic asthma symptoms. We induced allergic asthma by house dust mite (HDM) in A/J WT mice and Zpbp2 KO mice expressing lower levels of Ormdl3 mRNA than WT. We investigated the effect of a novel formulation of Fenretinide, LAU-7b, on the AHR, inflammatory cell infiltration, mucus production, IgE levels, and ceramide levels. Although lower Ormdl3 expression, which was observed in Zpbp2 KO mice, was associated with lower AHR, allergic Zpbp2 KO mice were not protected from inflammatory cell infiltration, mucus accumulation, or aberrant levels of VLCCs and LCCs induced by HDM. LAU-7b treatment protects both the Zpbp2 KO and WT mice. The treatment significantly lowers the gene expression of Ormdl3, normalizes the VLCCs and LCCs, and corrects all the other phenotypes associated with allergic asthma after HDM challenge, except the elevated levels of IgE. LAU-7b treatment prevents the augmentation of Ormdl3 expression and ceramide imbalance induced by HDM challenge and protects both WT and Zpbp2 KO mice against allergic asthma symptoms. SIGNIFICANCE STATEMENT: Compared with A/J WT mice, KO mice with Zpbp2 gene deletion have lower AHR and lower levels of Ormdl3 expression. The novel oral clinical formulation of Fenretinide (LAU-7b) effectively lowers the AHR and protects against inflammatory cell infiltration and mucus accumulation induced by house dust mite in both Zpbp2 KO and WT A/J mice. LAU-7b prevents Ormdl3 overexpression in WT allergic mice and corrects the aberrant levels of very-long-chain and long-chain ceramides in both WT and Zpbp2 KO allergic mice.

Sex biases in the genome-wide distribution of DNA methylation and gene expression levels are some of the manifestations of sexual dimorphism in mammals. To advance our understanding of the mechanisms that contribute to sex biases in DNA methylation and gene expression, we conducted whole genome bisulfite sequencing (WGBS) as well as RNA-seq on liver samples from mice with different combinations of sex phenotype and sex-chromosome complement. We compared groups of animals with different sex phenotypes, but the same genetic sexes, and vice versa, same sex phenotypes, but different sex-chromosome complements. We also compared sex-biased DNA methylation in mouse and human livers. Our data show that sex phenotype, X-chromosome dosage, and the presence of Y chromosome shape the differences in DNA methylation between males and females. We also demonstrate that sex bias in autosomal methylation is associated with sex bias in gene expression, whereas X-chromosome dosage-dependent methylation differences are not, as expected for a dosage-compensation mechanism. Furthermore, we find partial conservation between the repertoires of mouse and human genes that are associated with sex-biased methylation, an indication that gene function is likely to be an important factor in this phenomenon.

Zona pellucida binding protein 2 (

Genome-wide association study (GWAS) loci for several immunity-mediated diseases (early onset asthma, inflammatory bowel disease (IBD), primary biliary cholangitis, and rheumatoid arthritis) map to chromosomal region 17q12-q21. The predominant view is that association between 17q12-q21 alleles and increased risk of developing asthma or IBD is due to regulatory variants. ORM sphingolipid biosynthesis regulator (ORMDL3) residing in this region is the most promising gene candidate for explaining association with disease. However, the relationship between 17q12-q21 alleles and disease is complex suggesting contributions from other factors, such as trans-acting genetic and environmental modifiers or circadian rhythms. Circadian rhythms regulate expression levels of thousands of genes and their dysregulation is implicated in the etiology of several common chronic inflammatory diseases. However, their role in the regulation of the 17q12-q21 genes has not been investigated. Moreover, the core clock gene nuclear receptor subfamily 1, group D, member 1 (NR1D1) resides about 200 kb distal to the GWAS region. We hypothesized that circadian rhythms influenced gene expression levels in 17q12-q21 region and conversely, regulatory elements in this region influenced transcription of the core clock gene NR1D1 in cis. To test these hypotheses, we examined the diurnal expression profiles of zona pellucida binding protein 2 (ZPBP2/Zpbp2), gasdermin B (GSDMB), and ORMDL3/Ormdl3 in human and mouse tissues and analyzed the impact of genetic variation in the ZPBP2/Zpbp2 region on NR1D1/Nr1d1 expression. We found that Ormdl3 and Zpbp2 were controlled by the circadian clock in a tissue-specific fashion. We also report that deletion of the Zpbp2 region altered the expression profile of Nr1d1 in lungs and ileum in a time-dependent manner. In liver, the deletion was associated with enhanced expression of Ormdl3. We provide the first evidence that disease-associated genes Zpbp2 and Ormdl3 are regulated by circadian rhythms and the Zpbp2 region influences expression of the core clock gene Nr1d1.

Heterozygosity for Robertsonian translocations hampers pairing and synapsis between the translocated chromosome and its normal homologs during meiotic prophase I. This causes meiotic silencing of unsynapsed chromatin in pericentromeric regions. Several lines of evidence suggest that autosomal asynapsis leads to meiotic arrest in males and two underlying mechanisms have been proposed: (1) reactivation of the X and Y chromosomes due to competition for silencing factors and (2) meiotic silencing of genes that are located in the unsynapsed regions and are essential for meiotic progression. The latter mechanism requires that asynapsis and meiotic silencing spread beyond the p-arms of the normal homologs into gene-rich regions. We used chromatin immunoprecipitation assays to determine whether histones γH2AFX and H3.3, both marks of asynapsis and meiotic silencing, are enriched in gene-rich regions of the translocated chromosomes and their homologs in the spermatocytes of heterozygous carriers of Robertsonian translocations. We also asked if γH2AFX and H3.3 enrichment was reduced at the X chromosome and if γH2AFX and H3.3 enrichment was higher on the normal homolog. Our data show that γH2AFX enrichment extends as far as 9-15 Mb of the annotated genomic sequence of the q-arms of the translocated chromosomal trivalents and that both γH2AFX and H3.3 levels are reduced over the X chromosome. Our data are also suggestive of an asymmetry in γH2AFX and H3.3 enrichment with a bias toward the non-translocated homolog.

Background Sexual dimorphism in DNA methylation levels is a recurrent epigenetic feature in different human cell types and has been implicated in predisposition to disease, such as psychiatric and autoimmune disorders. To elucidate the genetic origins of sex-specific DNA methylation, we examined DNA methylation levels in fibroblast cell lines and blood cells from individuals with different combinations of sex chromosome complements and sex phenotypes focusing on a single autosomal region––the differentially methylated region (DMR) in the promoter of the zona pellucida binding protein 2 (ZPBP2) as a reporter. Results Our data show that the presence of the sex determining region Y (SRY) was associated with lower methylation levels, whereas higher X chromosome dosage in the absence of SRY led to an increase in DNA methylation levels at the ZPBP2 DMR. We mapped the X-linked modifier of DNA methylation to the long arm of chromosome X (Xq13-q21) and tested the impact of mutations in the ATRX and RLIM genes, located in this region, on methylation levels. Neither ATRX nor RLIM mutations influenced ZPBP2 methylation in female carriers. Conclusions We conclude that sex-specific methylation differences at the autosomal locus result from interaction between a Y-linked factor SRY and at least one X-linked factor that acts in a dose-dependent manner. Electronic supplementary material The online version of this article (10.1186/s13293-018-0169-7) contains supplementary material, which is available to authorized users.

The human chromosomal region 17q12–q21 is one of the best replicated genome-wide association study loci for childhood asthma. The associated SNPs span a large genomic interval that includes several protein-coding genes. Here, we tested the hypothesis that the zona pellucida-binding protein 2 (ZPBP2) gene residing in this region contributes to asthma pathogenesis using a mouse model. We tested the lung phenotypes of knock-out (KO) mice that carry a deletion of the Zpbp2 gene. The deletion attenuated airway hypersensitivity (AHR) in female, but not male, mice in the absence of allergic sensitization. Analysis of the lipid profiles of their lungs showed that female, but not male, KO mice had significantly lower levels of sphingosine-1-phosphate (S1P), very long-chain ceramides (VLCCs), and higher levels of long-chain ceramides compared to wild-type controls. Furthermore, in females, lung resistance following methacholine challenge correlated with lung S1P levels (Pearson correlation coefficient 0.57) suggesting a link between reduced AHR in KO females, Zpbp2 deletion, and S1P level regulation. In livers, spleens and blood plasma, however, VLCC, S1P, and sphingosine levels were reduced in both KO females and males. We also find that the Zpbp2 deletion was associated with gain of methylation in the adjacent DNA regions. Thus, we demonstrate that the mouse ortholog of ZPBP2 has a role in controlling AHR in female mice. Our data also suggest that Zpbp2 may act through regulation of ceramide metabolism. These findings highlight the importance of phospholipid metabolism for sexual dimorphism in AHR.

Chromosomal region 17q12-q21 is associated with asthma and harbors regulatory polymorphisms that influence expression levels of all five protein-coding genes in the region: IKAROS family zinc finger 3 (Aiolos) (IKZF3), zona pellucida binding protein 2 (ZPBP2), ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), and gasdermins A and B (GSDMA, GSDMB). Furthermore, DNA methylation in this region has been implicated as a potential modifier of the genetic risk of asthma development. To further characterize the effect of DNA methylation, we examined the impact of treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) that causes DNA demethylation, on expression and promoter methylation of the five 17q12-q21 genes in the human airway epithelium cell line NuLi-1, embryonic kidney epithelium cell line 293T and human adenocarcinoma cell line MCF-7. 5-aza-dC treatment led to upregulation of expression of GSDMA in all three cell lines. ZPBP2 was upregulated in NuLi-1, but remained repressed in 293T and MCF-7 cells, whereas ORMDL3 was upregulated in 293T and MCF-7 cells, but not NuLi-1. Upregulation of ZPBP2 and GSDMA was accompanied by a decrease in promoter methylation. Moreover, 5-aza-dC treatment modified allelic expression of ZPBP2 and ORMDL3 suggesting that different alleles may respond differently to treatment. We also identified a polymorphic CTCF-binding site in intron 1 of ORMDL3 carrying a CG SNP rs4065275 and determined its methylation level. The site's methylation was unaffected by 5-aza-dC treatment in NuLi-1 cells. We conclude that modest changes (8-13%) in promoter methylation levels of ZPBP2 and GSDMA may cause substantial changes in RNA levels and that allelic expression of ZPBP2 and ORMDL3 is mediated by DNA methylation.

The Dio3 gene, which encodes for the type 3 deiodinase (D3), controls thyroid hormone (TH) availability. The lack of D3 in mice results in tissue overexposure to TH and a broad neuroendocrine phenotype. Dio3 is an imprinted gene, preferentially expressed from the paternally inherited allele in the mouse fetus. However, heterozygous mice with paternal inheritance of the inactivating Dio3 mutation exhibit an attenuated phenotype when compared with that of Dio3 null mice. To investigate this milder phenotype, the allelic expression of Dio3 was evaluated in different mouse tissues. Preferential allelic expression of Dio3 from the paternal allele was observed in fetal tissues and neonatal brain regions, whereas the biallelic Dio3 expression occurred in the developing eye, testes, and cerebellum and in the postnatal brain neocortex, which expresses a larger Dio3 mRNA transcript. The newborn hypothalamus manifests the highest degree of Dio3 expression from the paternal allele, compared with other brain regions, and preferential allelic expression of Dio3 in the brain relaxed in late neonatal life. A methylation analysis of two regulatory regions of the Dio3 imprinted domain revealed modest but significant differences between tissues, but these did not consistently correlate with the observed patterns of Dio3 allelic expression. Deletion of the Dio3 gene and promoter did not result in significant changes in the tissue-specific patterns of Dio3 allelic expression. These results suggest the existence of unidentified epigenetic determinants of tissue-specific Dio3 imprinting. The resulting variation in the Dio3 allelic expression between tissues likely explains the phenotypic variation that results from paternal Dio3 haploinsufficiency.

Chromosomal region 17q12-q21 is one of the best-replicated genome-wide association study (GWAS) hits and associated with childhood-onset asthma. However, the mechanism by which the genetic association is restricted to childhood-onset disease is unclear. During childhood, more boys than girls develop asthma. Therefore, we tested the hypothesis that the 17q12-q21 genetic association was sex-specific. Indeed, a TDT test showed that in the Saguenay-Lac-Saint-Jean familial collection, the 17q12-q21 association was significant among male, but not among female asthmatic subjects. We next hypothesized that the bias in the genetic association resulted from sex-specific and/or age-dependent DNA methylation at regulatory regions and determined the methylation profiles of five 17q12-q21 gene promoters using the bisulfite sequencing methylation assay. We identified a single regulatory region within the zona pellucida binding protein 2 (ZPBP2) gene, which showed statistically significant differences between males and females with respect to DNA methylation. DNA methylation also varied with age and was higher in adult males compared to boys. We have recently identified two functionally important polymorphisms, both within the ZPBP2 gene that influence expression levels of neighboring genes. Combined with the results of the present work, these data converge pointing to the same 5 kb region within the ZPBP2 gene as a critical region for both gene expression regulation and predisposition to asthma. Our data show that sex- and age-dependent DNA methylation may act as a modifier of genetic effects and influence the results of genetic association studies.

We recently observed a reliable phenotypic difference in the inflammatory pain sensitivity of a congenic mouse strain compared to its background strain. By constructing and testing subcongenic strains combined with gene-expression assays, we provide evidence for the candidacy of the Yy1 gene - encoding the ubiquitously expressed and multifunctional Yin Yang 1 transcription factor - as responsible. To confirm this hypothesis, we used a Cre/lox strategy to produce mutant mice in which Yy1 expression was ablated in Nav 1.8-positive neurons of the dorsal root ganglion. These mutants also displayed reduced inflammatory pain sensitivity on the formalin test. Further testing of pain-related phenotypes in these mutants revealed robustly increased sensitivity to systemic and spinal (but not supraspinal) morphine analgesia, and greatly increased endogenous (swim stress-induced) opioid analgesia. None of the known biological roles of Yin Yang 1 were suggestive of such a phenotype, and thus a novel player in pain modulatory systems has been identified.

Phenotypic variation results from variation in gene expression, which is modulated by genetic and/or epigenetic factors. To understand the molecular basis of human disease, interaction between genetic and epigenetic factors needs to be taken into account. The asthma-associated region 17q12-q21 harbors three genes, the zona pellucida binding protein 2 (ZPBP2), gasdermin B (GSDMB) and ORM1-like 3 (ORMDL3), that show allele-specific differences in expression levels in lymphoblastoid cell lines (LCLs) and CD4+ T cells. Here, we report a molecular dissection of allele-specific transcriptional regulation of the genes within the chromosomal region 17q12-q21 combining in vitro transfection, formaldehyde-assisted isolation of regulatory elements, chromatin immunoprecipitation and DNA methylation assays in LCLs. We found that a single nucleotide polymorphism rs4795397 influences the activity of ZPBP2 promoter in vitro in an allele-dependent fashion, and also leads to nucleosome repositioning on the asthma-associated allele. However, variable methylation of exon 1 of ZPBP2 masks the strong genetic effect on ZPBP2 promoter activity in LCLs. In contrast, the ORMDL3 promoter is fully unmethylated, which allows detection of genetic effects on its transcription. We conclude that the cis-regulatory effects on 17q12-q21 gene expression result from interaction between several regulatory polymorphisms and epigenetic factors within the cis-regulatory haplotype region. Electronic supplementary material The online version of this article (doi:10.1007/s00439-012-1142-x) contains supplementary material, which is available to authorized users.

Cis-acting variants altering gene expression are a source of phenotypic differences. The cis-acting components of expression variation can be identified through the mapping of differences in allelic expression (AE), which is the measure of relative expression between two allelic transcripts. We generated a map of AE associated SNPs using quantitative measurements of AE on Illumina Human1M BeadChips. In 53 lymphoblastoid cell lines derived from donors of European descent, we identified common cis variants affecting 30% (2935/9751) of the measured RefSeq transcripts at 0.001 permutation significance. The pervasive influence of cis-regulatory variants, which explain 50% of population variation in AE, extend to full-length transcripts and their isoforms as well as to unannotated transcripts. These strong effects facilitate fine mapping of cis-regulatory SNPs, as demonstrated by dissection of heritable control of transcripts in the systemic lupus erythematosus-associated C8orf13-BLK region in chromosome 8. The dense collection of associations will facilitate large-scale isolation of cis-regulatory SNPs.

Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes.

Transmission ratio distortion (TRD) is a deviation from the expected Mendelian 1:1 ratio of alleles transmitted from parents to offspring and may arise by different mechanisms. Earlier we described a grandparental-origin-dependent sex-of-offspring-specific TRD of maternal chromosome 12 alleles closely linked to an imprinted region and hypothesized that it resulted from imprint resetting errors in the maternal germline. Here, we report that the genotype of the parents for loss-of-function mutations in the Dnmt1 gene influences the transmission of grandparental chromosome 12 alleles. More specifically, maternal Dnmt1 mutations restore Mendelian transmission ratios of chromosome 12 alleles. Transmission of maternal alleles depends upon the presence of the Dnmt1 mutation in the mother rather than upon the Dnmt1 genotype of the offspring. Paternal transmission mirrors the maternal one: live-born offspring of wild-type fathers display 1:1 transmission ratios, whereas offspring of heterozygous Dnmt1 mutant fathers tend to inherit grandpaternal alleles. Analysis of allelic transmission in the homologous region of human chromosome 14q32 detected preferential transmission of alleles from the paternal grandfather to grandsons. Thus, parental Dnmt1 is a modifier of transmission of alleles at an unlinked chromosomal region and perhaps has a role in the genesis of TRD.

International audience; The functioning of the genome is tightly related to its architecture. Therefore, understanding the relationship between different regulatory mechanisms and the organization of chromosomal domains is essential for understanding genome regulation. The majority of imprinted genes are assembled into clusters, share common regulatory elements, and, hence, represent an attractive model for studies of regulation of clusters of non-paralogous genes. Here, we investigated the relationship between genomic imprinting and diurnal regulation of genes from the imprinted domain of mouse chromosome 12. We compared gene expression patterns in C57BL/6 mice and congenic mice that carry the imprinted region from a Mus musculus molossinus strain MOLF/Ei. In the C57BL/6 mice, a putative enhancer/oscillator regulated the expression of only Mico1/Mico1os, whereas in the congenic mice its influence was spread onto Rtl1as, Dio3 and Dio3os, i.e. the distal part of the imprinted domain, resulting in coordinated diurnal variation in expression of five genes. Using additional congenic strains we determined that in C57BL/6 the effect of the putative enhancer/oscillator was attenuated by a linked dominant trans-acting factor located in the distal portion of chromosome 12. Our data demonstrate that (i) in adult organs, mRNA levels of several imprinted genes vary during the day, (ii) genetic variation may remove constraints on the influence of an enhancer and lead to spreading of its effect onto neighboring genes, thereby generating genotype-dependent expression patterns and (iii) different regulatory mechanisms within the same domain act independently and do not seem to interfere with each other.

Background Two asthma-associated regions 17q12–q21 and 5q31.1 harbour genes that show strong effect of genotype on expression levels. DNA methylation has an important role in gene regulation; therefore, we examined DNA methylation at promoters of 12 genes from 5q31 and 17q12–q21 regions. Our goal was to determine whether DNA methylation was associated with predisposition to asthma and whether such a relationship was independent from genetic association. Methods Using sodium bisulfite sequencing and pyrosequencing methylation assays, we examined the effect of genotype on DNA methylation in peripheral blood cells from individuals from the Saguenay-Lac-Saint-Jean asthma familial collection and lymphoblastoid cell lines. Results The local genotype influenced methylation levels of solute carrier family 22 (organic 3 cation/carnitine transporter) member 5 ( SLC22A5 ), zona pellucida binding protein 2 ( ZPBP2 ) and gasdermin A ( GSDMA ) promoter regions. The genotype had a dominant effect on ZPBP2 and GSDMA methylation with lower methylation levels in individuals that carry the asthma-predisposing alleles. Males also had lower methylation at the ZPBP2 promoter than females. We did not observe an effect of asthma status that would be independent of the genotype and the sex effects in the GSDMA , ZPBP2 and SLC22A5 regions; however, GSDMA and ZPBP2 data were suggestive of interaction between asthma and methylation levels in females and SLC22A5 in males. Conclusions The local genotype influences methylation levels at SLC22A5 and ZPBP2 promoters independently of the asthma status. Further studies are necessary to confirm the relationship between GSDMA-ZPBP2 and SLC22A5 methylation and asthma in females and males separately.

The expression of six imprinted genes (Dlk1, Gt12, Igf2r, Kcnq1, Nnat, and Peg1) was examined in brains of 21 mice derived from N2 x N2 intercrosses between C57BL/6 and MOLF/Ei strains. Imprinting of Igf2r, Kcnq1, Gt12, and Dlk1 varied among individuals. As three of these genes are implicated in cell-cell signaling or cell-environment interactions, variation in their imprinting may influence a wide range of biological processes from cell differentiation to behavior. To elucidate the mechanisms underlying the interindividual imprinting variation in the brain, we focused our effort on the paternally expressed gene Dlk1. We investigated expression of Dlk1 in the brains of animals from N9 and N10 backcrosses and found that reactivation of the normally silent maternal Dlk1 allele in the N9 and N10 mice occurred less often than in N2 x N2 animals. Our data suggest that trans-acting genetic factors of MOLF/Ei origin facilitate the reactivation of the normally silent maternal allele of Dlk1. We mapped one of these factors to the proximal part of Chr 7. The results of bisulfite sequencing methylation analysis show that reactivation of the maternal allele was also associated with hypermethylation of the intragenic differentially methylated region (IG DMR), which is the imprinting control region for the Dlk1-Gt12 domain. Thus, the imprinting status of Dlk1 in the brain depends upon trans-acting genetic influences and correlates with the methylation status of a specific subregion of the IG DMR.

Summary: Defects in expression of imprinted genes are believed to cause developmental abnormalities and play a role in carcinogenesis. To determine whether spontaneous imprinting defects may occur in mouse embryos, we studied the expression of two imprinted genes H19 and Igf2 in individual postimplantation 7.5 d.p.c. and 8.5 d.p.c. embryos. Biallelic expression of H19 was found in 1.6% of the embryos, whereas biallelic expression of Igf2 was found in 0.5% of the embryos. The loss of H19 imprinting (LOI) observed in a small fraction of early postimplantation embryos may be purely stochastic. Alternatively, since we never observed it in an inbred background, it may depend on genetic factors acting in trans. Either mechanism could explain the occurrence of polymorphic imprinting as well as the genesis of sporadic imprinting defects, including cancer. The frequency of LOI of H19 was higher than the incidence of sporadic imprinting disorders in humans (about 1 in 20,000). This contradiction may be explained by different incidence of imprinting errors in different imprinted regions of the genome, in different species, or by loss of the majority of nonmosaic embryos with imprinting defects before birth. genesis 31:11–16, 2001. © 2001 Wiley-Liss, Inc.

The vitamin D binding protein/Gc-globulin (DBP) gene is a member of a multigene cluster that includes albumin (ALB), α-fetoprotein (AFP), and α-albumin/afamin (AFM). All four genes have structural and functional similarities and map to the same chromosomal regions in humans (4q11–q13), mice, and rats. An accurate physical map of the region encompassing these genes is a prerequisite for study of their respective transcriptional regulation and identification of potential shared regulatory elements. By refining the physical and meiotic maps of the 4q11–q13 region and creating a local PAC contig, the order and transcriptional orientations of these four genes were determined to be centromere–3′-DBP-5′–5′-ALB-3′–5′-AFP-3′–5′-AFM3′–telomere. The ancestral DBP gene was separated from the ALBgene by >1.5 Mb. This organization and spacing establishes a foundation for ongoing functional studies in this region.

We report on a female with mental and motor retardation, facial dysmorphism, abnormal pigmentation reminiscent to hypomelanosis of Ito (HI), and karyotypic mosaicism involving a small supernumerary marker chromosome. The marker chromosome was defined by fluorescence in situ hybridisation (FISH) as a ring X chromosome with breakpoints in the juxtacentromeric region. FISH analysis showed that the ring does not include the XIST locus at the X-inactivation centre and, therefore, may not be subject to X inactivation. X-inactivation studies with the HUMARA (human androgen receptor) and FMR1 assay showed a skewed X-inactivation pattern (85:15) with preferential inactivation of the paternal X chromosome. These results are discussed with respect to the role of functional disomy of Xp in the pathogenesis of HI.

Summary We have analyzed the transmission of maternal alleles at loci spanning the length of the X chromosome in 47 normal, genetic disease–free families. We found a significant deviation from the expected Mendelian 1:1 ratio of grandpaternal:grandmaternal alleles at loci in Xp11.4-p21.1. The distortion in inheritance ratio was found only among male offspring and was manifested as a strong bias in favor of the inheritance of the alleles of the maternal grandfather. We found no evidence for significant heterogeneity among the families, which implies that the major determinant involved in the generation of the non-Mendelian ratio is epigenetic. Our analysis of recombinant chromosomes inherited by male offspring indicates that an 11.6-cM interval on the short arm of the X chromosome, bounded by DXS538 and DXS7, contains an imprinted gene that affects the survival of male embryos.

Failure of homologous synapsis during meiotic prophase triggers transcriptional repression. Asynapsis of the X and Y chromosomes and their consequent silencing is essential for spermatogenesis. However, asynapsis of portions of autosomes in heterozygous translocation carriers may be detrimental for meiotic progression. In fact, a wide range of phenotypic outcomes from meiotic arrest to normal spermatogenesis have been described and the causes of such a variation remain elusive. To better understand the consequences of asynapsis in male carriers of Robertsonian translocations, we focused on the dynamics of recruitment of markers of asynapsis and meiotic silencing at unsynapsed autosomal trivalents in the spermatocytes of Robertsonian translocation carrier mice. Here we report that the enrichment of breast cancer 1 (BRCA1) and histone γH2AX at unsynapsed trivalents declines during the pachytene stage of meiosis and differs from that observed in the sex body. Furthermore, histone variant H3.3S31, which associates with the sex chromosomes in metaphase I/anaphase I spermatocytes, localizes to autosomes in 12% and 31% of nuclei from carriers of one and three translocations, respectively. These data suggest that the proportion of spermatocytes with markers of meiotic silencing of unsynapsed chromatin (MSUC) at trivalents depends on both, the stage of meiosis and the number of translocations. This may explain some of the variability in phenotypic outcomes associated with Robertsonian translocations. In addition our data suggest that the dynamics of response to asynapsis in Robertsonian translocations differs from the response to sex chromosomal asynapsis in the male germ line.

Part I: Epigenetic phenomena in the germ line and early embryonic development and their effects on the inheritance of genetic traits.- Epigenetic reprogramming in the mammalian germ line.- Establishment of tissue-specific epigenetic states during development.- X-chromosome Inactivation.- Cis- and trans-effects underlying polar overdominance at the callipyge locus.- Transgenerational epigenetic effects and complex inheritance patterns.- Autosomal monoallelic expression.- Part II: Epigenetic variation in health and disease.- Recurrent CNVs in the etiology of epigenetic neurodevelopmental disorders.- Impact of the early-life environment on the epigenome and behavioral development.- Interaction between genetics and epigenetics in cancer.- Part III: Impact of epigenetics on complex trait genetics and analysis.- Epigenetic variation, phenotypic heritability, and evolution.- Statistical Approaches for Detecting Transgenerational Genetic Effects in Humans.- Transmission ratio distortion: a neglected phenomenon with many consequences in genetic analysis and population genetics.- Epigenome Wide Association Studies: Potential insights into human disease.- Analytical Considerations for Epigenome-wide Association Scans of Complex Traits.- Index.

Mendel’s laws describing the inheritance of simple genetic traits are based on the assumption that genes are transmitted unchanged from parents to offspring. However, this assumption is not always correct as the mammalian genome undergoes massive epigenetic reprogramming during gametogenesis and early embryonic development. Furthermore, stochastic or environmentally induced epigenetic variation may lead to situations where the epigenetic marking of the same allele differs between parents and offspring, among siblings or even monozygotic twins. Epigenetic marks may modify the penetrance of a phenotype and cause “non-Mendelian” inheritance even when the causal genetic variant is transmitted from parent to offspring in perfect Mendelian proportions. In this chapter we focus on a particular type of inheritance where epigenetic marks that are present in parental somatic cells fail to be reset in a proportion of parental germ cells and are transmitted to offspring. Such a transgenerational “epigenetic memory” increases the complexity of the inheritance pattern and therefore is of particular interest for geneticists.

The mammalian SR-type splicing factors ASF/SF2 and SC-35 play crucial roles in pre-mRNA splicing and have been shown to shift splice site choice in vitro. We have mapped the ASF/SF2 gene in mice and humans and the SC-35 gene in mice. Somatic cell hybrid mapping of the human ASF/SF2 gene (SFRS1 locus) reveals that it resides on chromosome 17, and fluorescence in situ hybridization refines this localization to 17q21.3-q22. Recombinant inbred mapping of the mouse ASF/SF2 gene (Sfrs1 locus) and the mouse SC-35 gene (Sfrs2 locus) demonstrates that both genes are located in a part of mouse chromosome 11 that is homologous to human chromosome 17. Mapping of Sfrs1 using F1 hybrid backcross mice between the strains C57BL/6 and DDK places Sfrs1 very near the marker D11Mit38 and indicates that the ASF/SF2 gene is closely linked to the Ovum mutant locus.

DNA methylation and DNA methyltransferases are essential for spermatogenesis. Mutations in the DNA methyltransferase Dnmt1 gene exert a paternal effect on epigenetic states and phenotypes of offspring, suggesting that DNMT1 is important for the epigenetic remodeling of the genome that takes place during spermatogenesis. However, the specific role of DNMT1 in spermatogenesis and the establishment of genomic imprints in the male germ line remains elusive. To further characterize the effect of DNMT1 deficiency on the resetting of methylation imprints during spermatogenesis, we analyzed the methylation profiles of imprinted regions in the spermatozoa of mice that were heterozygous for a Dnmt1 loss-of-function mutation. The mutation did not affect the H19 or IG differentially methylated regions (DMRs) that are usually highly methylated but led to a partial hypermethylation of the Snrpn DMR, a region that should normally be unmethylated in mature spermatozoa. This defect does not appear in mouse models with mutations in Dnmt3a and Mthfr genes and, therefore, it is specific for the Dnmt1 gene and is suggestive of a role of DNMT1 in imprint resetting or maintenance in the male germ line.

Preferential amplification of the paternal allele of the N–myc gene in human neuroblastomas

Meiotic silencing of unsynapsed chromatin (MSUC) occurs in the germ cells of translocation carriers and may cause meiotic arrest and infertility. We hypothesized that if bypassing meiotic checkpoints MSUC may cause epigenetic defects in sperm. We investigated the meiotic behavior of the Robertsonian translocation Rb (8.12) in mice. The unsynapsed 8 and 12 trivalent was associated with the XY body during early and mid-pachynema in heterozygous Rb (8.12) carriers, suggesting possible silencing of pericentromeric genes, such as the Dnmt3a gene. In wild-type mice, DNMT3A protein showed a dramatic accumulation in the nucleus during the mid-pachytene stage and distinct association with the XY body. In translocation carriers, DNMT3A was less abundant in a proportion of pachytene spermatocytes that also had unsynapsed pericentromeric regions of chromosomes 8 and 12. The same mice had incomplete methylation of the imprinted H19 differentially methylated region (DMR) in sperm. We propose that impaired H19 imprint establishment results from lack of synapsis in chromosomes 8 and 12 probably through transient silencing of a chromosome 8 or 12 gene during pachynema. Furthermore, our findings support the notion that imprint establishment at the H19 locus extends into pachynema.

DNA methylation patterns are often poorly conserved through cell culturing. To determine the effect of cell immortalization and culture on DNA methylation profiles, we analyzed methylation in the differentially methylated regions (DMR) of five imprinted domains: the intergenic (IG) DMR on chromosome 14q32; potassium voltage-gated channel, KQT-like subfamily, member 1, (KCNQ1); small nuclear ribonucleoprotein polypeptide N (SNRPN), mesoderm specific transcript homolog (MEST); and H19 in lymphoblastoid cell lines (LCLs). In the IG DMR we found an aberrant methylation pattern that was consistent through all the cell lines tested and significantly different from that of noncultured peripheral blood cells. Using a generalized linear mixed model to compare methylation profiles, we show that recently derived LCLs significantly differ from the CEPH LCLs. This implies a gradual cell-culture related deterioration of DNA methylation in the IG DMR with at least two steps that may be identified: loss of methylation at CG sites 1 and 8; and loss of allelic differences in DNA methylation. The IG DMR methylation profile also confirms the high level of clonality of the CEPH LCLs. We conclude that non-transformed primary cells may be less susceptible to epigenetic anomalies and therefore may provide a more accurate reflection of gene expression in vivo.

In an attempt to identify new genes responsible for variability in pain sensitivity, we tested three congenic mouse strains--in which a small portion of the genome of the MOLF/Ei strain has been placed on a C57BL/6 genetic background--on a battery of nine nociceptive assays, chosen to reflect those assays in most common use in the pain literature. Mice of both sexes were evaluated by two different testers at different points in time, allowing us to examine the relative importance of genotype, sex, tester and cohort effects on data from these assays. We find strong evidence for the existence of two quantitative trait loci (i.e., genomic regions containing variability-causing genes), one for thermal nociception on mouse chromosome (Chr) 17 (Chr 17; Tpnr3) and one for formalin test nociception on mouse Chr 12 (Nociq3). We note, however, that the nociceptive assays in this battery feature strong main effects and interactions of sex, tester, and cohort, which if not controlled or covaried can seriously confound interpretation of genetic experiments, including the comparison of transgenic knockout mice to their wild-type controls.

One form of myotonic dystrophy, dystrophia myotonica 1 (DM1), is caused by the expansion of a (CTG)(n) repeat within the dystrophia myotonica-protein kinase (DMPK) gene located in chromosome region 19q13.3. Unaffected individuals carry alleles with repeat size (CTG)(5-37), premutation carriers (CTG)(38-49) and DM1 affected individuals (CTG)(50-6,000). Preferential transmission both of expanded repeats from DM1-affected parents and larger DMPK alleles in the normal-size range have been reported in live-born offspring. To determine the moment in development when transmission ratio distortion (TRD) for larger normal-size DMPK alleles is generated, the transmission from heterozygous parents with one repeat within the (CTG)(5-18) range (Group I repeat) and the other within the (CTG)(19-37) range (Group II repeat) to human preimplantation embryos was analysed. A statistically significant TRD of 59% (95% confidence interval of 54-64) in favour of Group II repeats from both mothers and fathers was observed in preimplantation embryos, which remained significant when female embryos were considered separately. In contrast, no significant TRD was detected for repeats from informative Group I/Group I parents. Our analysis showed that Group II repeats specifically were preferentially transmitted in human preimplantation embryos. We suggest that TRD, in Group II repeats at the DMPK locus, is likely to result from events occurring at or around the time of fertilisation.

To investigate the genetic influence on X chromosome inactivation and on age-related skewing of X inactivation, in particular, we analysed the X inactivation pattern (XIP) in peripheral blood cells from 118 young monozygotic (MZ) twin pairs (18–53 years), 82 elderly MZ twin pairs (55–94 years), 146 young dizygotic (DZ) twin pairs (20–54 years) and 112 elderly DZ twin pairs (64–95 years). Elderly twins had a higher frequency of skewed X inactivation (34%) than young twins (15%) (P

We investigated the effect of aging on X chromosome inactivation by performing a longitudinal study in a population of 178 normal females. We examined X-inactivation ratios (fraction of cells with the same X chromosome active) in two sets of peripheral blood DNA samples collected about two decades apart. We observed a strong correlation between the ratios of individual females at the two time points and found no significant difference between the two sets of measurements. These observations indicate that aging, per se (as opposed to being “aged”), has little effect on X-inactivation. However, we also found that several females who were older than 60 years of age at the time of the first measurement acquired significant changes in the X-inactivation ratio. We speculate that, if X-inactivation skewing is a frequently acquired trait in older females, it is acquired as the result of a discontinuous or catastrophic process and is not the result of constant selection for or against hematopoietic stem cells with a particular X chromosome active.

The identification of human sequence polymorphisms that regulate gene expression is key to understanding human genetic diseases. We report a survey of human genes that demonstrate allelic differences in gene expression, reflecting the presence of putative allele-specific cis-acting factors of either genetic or epigenetic nature. The expression of allelic transcripts in heterozygous samples is assessed directly by relative quantitation of intragenic marker alleles in messenger or heteronuclear RNA derived from cells or tissues. This survey used 193 single-nucleotide polymorphisms (SNPs) from 129 genes expressed in lymphoblastoid cell lines, to identify 23 genes (18%) with common allele-specific transcripts whose expression deviated from the expected equimolar ratio. A subset of these deviations, or “allelic imbalances,” can be observed in multiple samples derived from reference CEPH (“Centre d’Etude du Polymorphisme Humain”) pedigrees and demonstrate a spectrum of patterns of transmission, including cosegregation of allelic skewing across generations compatible with Mendelian inheritance as well as random monoallelic expression for three genes ( IL1A, HTR2A, and FGB). Additional studies for BTN3A2 provide evidence of SNPs and haplotypes in complete linkage disequilibrium with high- and low-expressing transcripts. The pipeline described herein offers tools for efficient identification and characterization of allelic expression allowing identification of regulatory sequence variants as well as epigenetic variation affecting human gene expression.

Deviations from Mendelian 1:1 transmission ratio have been observed in mice and humans. With few exceptions, the mechanism leading to transmission-ratio distortion (TRD) remains obscure. We proposed that a genomic imprinting mechanism plays a key role in the genesis of grandparental origin-dependent TRD (Naumova et al. 2001). To further test this hypothesis, we analyzed the transmission of grandparental alleles at three imprinted regions of the mouse genome known to contain genes required for embryo development. We found and replicated moderate (58%: 42%) TRD in favor of grandmaternal alleles in the imprinted region of maternal distal Chromosome (Chr) 12 among female offspring. Comparison of transmission ratios at the distorted region of Chr 12 among 3-week-old mice with those in embryos suggests that the distortion in favor of grandmaternal alleles is owing to postimplantation embryo loss. The absence of grandparental origin-dependent TRD for maternal Chr 6 and 7 implies that the relationship between TRD and imprinting is complex. Most likely, multiple conditions are required for TRD to occur.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease caused by mutations in the arginine vasopressin receptor 2 gene (AVPR2). Thirty-three novel AVPR2 mutations were identified in 62 families that were not included in our previous studies. This study describes the diversity of mutations observed in a total of 117 families, the number of affected people at the time of diagnosis, skewed X chromosome inactivation in severely affected females, the inferred parental origin of de novo mutations, and it provides estimates of incidence. Among 117 families, there were 82 different putative disease-causing mutations. Based on haplotype analysis, it can be inferred that when the same AVPR2 mutation is identified in different families that were not known to be related, the mutations most likely arose independently. More than half of the families had only one affected male; two families presented with a severely affected female and no family history of NDI. A de novo mutation arose during oogenesis in the mother in 20% of isolated cases. The estimate of about 8.8 per million male live births of the incidence of X-linked NDI in the province of Quebec, Canada may be representative of the general population except in Nova Scotia and New Brunswick, where the incidence is more than six times higher. Documentation of the diversity of mutations will assist in revealing the full spectrum of clinical variation. Discussion of genetic and population genetic aspects of X-linked NDI may contribute to early diagnosis and treatment.