Imprinting mutations suggested by abnormal DNA methylation patterns in familial Angelman and Prader-Willi syndromes.

The D15S9 and D15S63 loci in the Prader-Willi/Angelman syndrome region on chromosome 15 are subject to parent-of-origin-specific DNA methylation. We have found two Prader-Willi syndrome families in which the patients carry a maternal methylation imprint on the paternal chromosome. In one of these families, the patients have a small deletion encompassing the gene for the small nuclear ribonucleoprotein polypeptide N, which maps 130 kb telomeric to D15S63. Furthermore, we have identified a pair of nondeletion Angelman syndrome sibs and two isolated Angelman syndrome patients who carry a paternal methylation imprint on the maternal chromosome. These Angelman and Prader-Willi syndrome patients may have a defect in the imprinting process in 15q11-13. We propose a model in which a cis-acting mutation prevents the resetting of the imprinting signal in the germ line and thus disturbs the expression of imprinted genes in this region.     

A genetic model for the Prader-Willi syndrome and its implication for angelman syndrome

Summary Sporadic cases of Prader-Willi syndrome (PWS) are associated with the physical absence of the paternal Prader-Willi chromosome region (PWCR) by deletion 15q11–13, by segmental maternal heterodisomy or by chromosome rearrangements resulting in homozygosity for maternal PWCR. In isolated/familial cases, it is proposed that the expression of PWS depends on the functional absence caused by mutated gene(s) within the paternal PWCR. The same mutation on a maternally derived chromosome 15 is not able to express PWS. An epigenetic mechanism associated with the paternal meiosis is essential. In the Angelman syndrome (AS), inverse mechanisms are postulated. There is convincing evidence for specific PWS and AS genes or alleles within PWCR. This is compatible with the observations of interstitial chromosome deletions of the critical region in normal individuals or in probands with phenotypes other than PWS or AS. The new ideas of the model stated here are: (1) the proposed epigenetic mechanism in PWCR is obviously common in humans, but is usually of no phenotypic relevance; (2) interactions with specific chromosomal or gene mutations are required for the clinical expression of PWS or AS; (3) each factor alone is not able to produce an abnormal phenotype.     

Robust, easy, and dose-sensitive methylation test for the diagnosis of Prader-Willi and Angelman syndromes

We present a new method for differential diagnosis of Prader-Willi (PWS) and Angelman syndromes (AS) that requires only a small amount of DNA including that obtained from amniocentesis specimens. This method not only proved to be robust and rapid, but, most importantly, it can be dosage sensitive, supplying additional information useful for genetic counselling. After methylation-dependent digestion of DNA with HpaII or McrBC, exon 1 of the SNRPN gene is amplified together with a sequence in the CpG island of the H19 gene. Given the similarities in sequence composition and methylation status between the amplified sequences, their co-amplification under semiquantitative conditions allows an easy discrimination between single dosage (present in deletions or chromosomal translocations) and a double-dosage state (uniparental disomy or imprinting error), when the appropriate controls are included. The method we have developed in combination with standard cytogenetic studies and segregation analysis of microsatellite markers offers a rapid and easy procedure to resolve most suspected cases of PWS and AS, and consequently to provide accurate genetic counselling.     

Molecular diagnosis of the Prader-Willi and Angelman syndromes by detection of parent-of-origin specific DNA methylation in 15q11-13

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are distinct genetic disorders that are caused by a deletion of chromosome region 15q11-13 or by uniparental disomy for chromosome 15. Whereas PWS results from the absence of a paternal copy of 15q11-13, the absence of a maternal copy of 15q11-13 leads to AS. We have found that an MspI/HpaII restriction site at the D15S63 locus in 15q11-13 is methylated on the maternally derived chromosome, but unmethylated on the paternally derived chromosome. Based on this difference, we have devised a rapid diagnostic test for patients suspected of having PWS and AS.     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.     

DNA methylation profiling distinguishes histological subtypes of renal cell carcinoma

Renal cell carcinoma (RCC) accounts for around 3% of cancers in the UK, and both incidence and mortality are increasing with the aging population. RCC can be divided into several subtypes: conventional RCC (the most common, comprising 75% of all cases), papillary RCC (15%) and chromophobe RCC (5%). Renal oncocytoma is a benign tumor and accounts for 5% of RCC. Cancer and epigenetics are closely associated, with DNA hypermethylation being widely accepted as a feature of many cancers. In this study the DNA methylation profiles of chromophobe RCC and renal oncocytomas were investigated by utilizing the Infinium HumanMethylation450 BeadChips. Cancer-specific hypermethylation was identified in 9.4% and 5.2% of loci in chromophobe RCC and renal oncocytoma samples, respectively, while the majority of the genome was hypomethylated. Thirty (hypermethylated) and 41 (hypomethylated) genes were identified as differentially methylated between chromophobe RCC and renal oncocytomas (p < 0.05). Pathway analysis identified some of the differentially hypermethylated genes to be involved in Wnt (EN2), MAPK (CACNG7) and TGFβ (AMH) signaling, Hippo pathway (NPHP4), and cell death and apoptosis (SPG20, NKX6-2, PAX3 and BAG2). In addition, we analyzed ccRCC and papillary RCC data available from The Cancer Genome Atlas portal to identify differentially methylated loci in chromophobe RCC and renal oncocytoma in relation to the other histological subtypes, providing insight into the pathology of RCC subtypes and classification of renal tumors.     

Bisphenol A exposure modifies DNA methylation of imprint genes in mouse fetal germ cells

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Human frequent exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on DNA methylation of imprinting genes in fetal mouse germ cell. Pregnant mice were treated with BPA at doses of 0, 40, 80 and 160 μg BPA/kg body weight/day from 0.5 day post coitum. DNA methylation of imprinting genes, Igf2r, Peg3 and H19, was decreased with the increase of BPA concentration in fetal mouse germ cells (p < 0.01).The relative mRNA levels of Nobox were lower in BPA-treated group compared to control (BPA free) in female fetal germ cells, but in male fetal germ cells, a significant higher in Nobox expression was observed in BPA-treated group compared to control. Decreased mRNA expression of specific meiotic genes including Stimulated by Stra8 and Dazl were obtained in the female fetal germ cells. In conclusion, BPA exposure can affect the DNA methylation of imprinting genes in fetal mouse germ cells.     

DNA methylation profiling distinguishes three clusters of breast cancer cell lines

Methylation change plays an important role in many cellular systems, including cancer development. During recent years, genome-wide or large-scale methylation data has become available thanks to rapid advances in high-throughput biotechnologies. So far, researchers have always used gene expression profiling to study disease subtypes and related therapies. In this study, we investigated methylation profiles in 30 breast cancer cell lines using methylation data generated by microarray technologies. Strong variation of the number of methylation peaks was found among these 30 cell lines; however, more peaks were found in the upstream regions than in downstream regions of genes. We further grouped the methylation profiles of these cell lines into three consensus clusters. Finally, we performed an integrative analysis of breast cancer cell lines using both methylation and gene-expression profiling data. There was no significant correlation between methylation-profiling subtypes and gene-expression profiling subtypes, suggesting the complex nature of methylation in the regulation of gene expression. However, we found basal B cell lines appeared exclusively in two methylation clusters. Although these results are preliminary, this study suggests that methylation profiling might be promising in disease subtype classification and the development of therapeutic strategies.     

Evaluation of methylation analysis for diagnostic testing in 258 referrals suspected of Prader-Willi or Angelman syndromes

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurodevelopmental disorders with interrelated genetic mechanisms because genomic imprinting within the chromosome 15q11–13 region affects both the PWS and the AS locus. Methylation analysis is one method of distinguishing between the maternally and paternally inherited chromosome 15. Here we present clinical and molecular data on a large series of 258 referred patients, evaluated with methylation analysis: 115 with suspected PWS and 143 with suspected AS. In these patients, the clinical phenotype was graded into three groups: classical (group 1); not classical but possible (group 2); not classical and unlikely (group 3). For PWS, a fourth group consisted of hypotonic babies. DNA methylation analysis confirmed the diagnosis of PWS in 30 patients (26%) and AS in 28 patients (20%). For 21 PWS patients the mechanism was established: 15 had deletions, 4 had uniparental disomy (UPD) and 2 a presumed imprinting defect. Clinically all those with an abnormal methylation pattern had the classical phenotype and none of those with a normal methylation pattern had classical PWS. For 23 AS patients in whom a mechanism was established, 17 had a deletion, 3 had UPD and 3 had a presumed imprinting defect. There was greater clinical overlap in AS, with 26 classical AS patients having a normal methylation pattern while an abnormal methylation pattern was seen in one patient from group 2. In addition, there were a further 40 patients with a normal methylation pattern in whom AS was still a possible diagnosis. Our conclusion is that methylation analysis provides an excellent screening test for both syndromes, providing ∼99% diagnosis for PWS and for AS, a 75% diagnostic rate, supplemented for the remaining 25% with an essential basic starting point to further investigations. Received: 10 February 1998 / Accepted: 7 July 1998     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ～3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ～2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ～750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Associations of sex hormone-binding globulin and testosterone with genome-wide DNA methylation

Background

Levels of sex hormone-binding globulin (SHBG) and the androgen testosterone have been associated with risk of diseases throughout the lifecourse. Although both SHBG and testosterone have been shown to be highly heritable, only a fraction of that heritability has been explained by genetic studies. Epigenetic modifications such as DNA methylation may explain some of the missing heritability and could potentially inform biological knowledge of endocrine disease mechanisms involved in development of later life disease. Using data from the Avon Longitudinal Study of Parents and Children (ALSPAC), we explored cross-sectional associations of SHBG, total testosterone and bioavailable testosterone in childhood (males only) and adolescence (both males and females) with genome-wide DNA methylation. We also report associations of a SHBG polymorphism (rs12150660) with DNA methylation, which leads to differential levels of SHBG in carriers, as a genetic proxy of circulating SHBG levels.

Results

We identified several novel sites and genomic regions where levels of SHBG, total testosterone, and bioavailable testosterone were associated with DNA methylation, including one region associated with total testosterone in males (annotated to the KLHL31 gene) in both childhood and adolescence and a second region associated with bioavailable testosterone (annotated to the CMYA5 gene) at both time-points. We also identified one region where both SHBG and bioavailable testosterone in males in childhood (annotated to the ZNF718 gene) was associated with DNA methylation.

Conclusion

Our findings have important implications in the understanding of the biological processes of SHBG and testosterone, with the potential for future work to determine the molecular mechanisms that could underpin these associations.

     

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.     

DNA methylation in mouse A-repeats in DNA methyltransferase-knockout ES cells and in normal cells determined by bisulfite genomic sequencing

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.     

DNA diagnosis of Prader-Willi and Angelman syndromes with the probe PW71 (D15S63)

Previously, 158 nuclear families with probands suspected of having either Prader Willi (PWS) or Angelman syndrome (AS) were analyzed with polymorphic DNA markers from the 15q11–13 region. These cases have been re-evaluated with the probe PW71 (D15S63), which detects parent-of-origin-specific alleles after digestion with a methylation-sensitive restriction enzyme (HpaII). Application of PW71 to DNA samples isolated from leucocytes, confirmed the deletions and uniparental disomies detected earlier by marker analysis, and resolved 50% of the previously uninformative (n=18) cases. PW71 and restriction fragment length polymorphism analysis indicated that, in all resolved cases, disomies of the 15q11–13 region were present. The use of PW71 increased the percentage of disomies detected in our PWS and AS patient groups. Almost 50% of our PWS patients and 17% of the AS patients showed a disomy of maternal or paternal origin, respectively. DNA of first trimester chorionic villi and of fibroblast cultures was not suitable for analysis with PW71 because of different methylation patterns. The application of PW71 is recommended for the diagnosis of the PWS and AS, with respect to DNA samples from blood.