Bisulphite sequencing of the transient neonatal diabetes mellitus DMR facilitates a novel diagnostic test but reveals no methylation anomalies in patients of unknown aetiology

Transient neonatal diabetes mellitus (TNDM) is associated with overexpression of an imprinted locus on chromosome 6q24; this locus contains a differentially methylated region (DMR) consisting of an imprinted CpG island that normally allows expression only from the paternal allele of genes under its control. Three types of abnormality involving 6q24 are known to cause TNDM: paternal uniparental disomy of chromosome 6 (pUPD6), an isolated methylation defect of the imprinted CpG island at chromosome 6q24 and a duplication of 6q24 of paternal origin. A fourth group of patients has no identifiable anomaly of 6q24. Bisulphite sequencing of the DMR has facilitated the development of a diagnostic test for TNDM based on ratiometric methylation-specific polymerase chain reaction. We have applied this method to 45 cases of TNDM, including 12 with pUPD6, 11 with an isolated methylation mutation at 6q24, 16 with a duplication of 6q24 and six of unknown aetiology, together with 29 normal controls. All were correctly assigned. The method is therefore capable of detecting all known genetic causes of TNDM at 6q24, although pUPD6 and methylation mutation cases are not distinguished from one another. In addition, we have carried out bisulphite sequencing of the DMR to compare its methylation status between six TNDM patients with a known methylation mutation, six patients with no identifiable 6q24 mutation and six normal controls. Whereas methylation mutation patients showed a near-total absence of DNA methylation at the TNDM locus, the patients with no identified molecular anomaly showed no marked methylation variation from controls.     

Maternal effect for DNA mismatch repair in the mouse.

DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was approximately 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.     

Transienter neonataler Diabetes und Hypomethylierungssyndrome

Transient neonatal diabetes (TNDM) is manifested before the age of 6 weeks and typically resolves within 18 months. Main clinical features include intrauterine growth retardation, hyperglycemia and dehydration with absent ketoacidosis. Causes of TNDM are heterogeneous but 70% are due to a chromosomal aberration in the region 6q24 which contains the imprinted genes PLAGL1/ZAC and HYMAI. Paternal uniparental disomy 6 (upd(6)pat) or paternal duplications of the imprinted region as well as imprinting defects of the maternal allele all result in an overexpression of the paternally expressed gene PLAGL1. Imprinting defects in 6q24 can occur as isolated events or can affect more than one locus (hypomethylation syndrome). Hypomethylation at multiple loci has so far been observed in patients with TNDM, Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS).The risk of recurrence depends on the underlying cause of TNDM. Chromosomal aberrations in the parents affecting chromosome 6 increase the risk for UPD or duplication of the imprinted locus in 6q24. Nevertheless, UPD and duplication 6q24 are mostly de novo occurrences.     

Identification of imprinting regulators at the Meg3 differentially methylated region

Genomic imprinting at the Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos.     

Mechanisms of Igf2/H19 imprinting: DNA methylation, chromatin and long-distance gene regulation

The mouse Igf2 and H19 genes lie 70-kb apart on chromosome 7 and are reciprocally imprinted. Two regulatory regions are important for their parental allele-specific expression: a differentially methylated region (DMR) upstream of H19 and a set of tissue-specific enhancers downstream of H19. The enhancers specifically activate Igf2 on the paternal chromosome and H19 on the maternal chromosome. The interactions between the enhancers and the genes are regulated by the DMR, which works as a selector by exerting dual functions: a methylated DMR on the paternal chromosome inactivates adjacent H19 and an unmethylated DMR on the maternal chromosome insulates Igf2 from the enhancers. These processes appear to involve methyl-CpG-binding proteins, histone deacetylases and the formation of chromatin insulator complexes. The Igf2/H19 region provides a unique model in which to study the roles of DNA methylation and chromatin structure in the regulation of chromosome domains.     

Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.     

A maternal hypomethylation syndrome presenting as transient neonatal diabetes mellitus

The expression of imprinted genes is mediated by allele-specific epigenetic modification of genomic DNA and chromatin, including parent of origin-specific DNA methylation. Dysregulation of these genes causes a range of disorders affecting pre- and post-natal growth and neurological function. We investigated a cohort of 12 patients with transient neonatal diabetes whose disease was caused by loss of maternal methylation at the TNDM locus. We found that six of these patients showed a spectrum of methylation loss, mosaic with respect to the extent of the methylation loss, the tissues affected and the genetic loci involved. Five maternally methylated loci were affected, while one maternally methylated and two paternally methylated loci were spared. These patients had higher birth weight and were more phenotypically diverse than other TNDM patients with different aetiologies, presumably reflecting the influence of dysregulation of multiple imprinted genes. We propose the existence of a maternal hypomethylation syndrome, and therefore suggest that any patient with methylation loss at one maternally-methylated locus may also manifest methylation loss at other loci, potentially complicating or even confounding the clinical presentation.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Altered anteroposterior polarity of micropyle ring formation in the eggs of Pyrrhocoris apterus L. (Heteroptera : Pyrrhocoridae)

The anterior pole of the normal eggs of Pyrrhocoris apterus (Heteroptera : Pyrrhocoridae) carries a ring of aeromicropyles. In DMR (double micropyle ring) eggs, an additional aeromicropyle ring occurs on the posterior egg pole. The number of micropyles forming this supernumerary ring varies, being usually considerably lower than that observed in the normal (anterior) ring. More than 6% of eggs laid by the Apricot (Ap) mutant of P. apterus are of the DMR type. Reciprocal crosses between the wild-type and Ap homozygous bugs demonstrate a maternal effect of the Ap mutation on the DMR egg production. Since DMR eggs are segregated at a low frequency in the wild-type strains as well as in all analyzed mutations of the bug, the DMR trait is most probably not primarily coded by the Ap gene. Physiological mechanisms involved in the formation of DMR eggs and the correlations between altered egg shell morphology and embryogenesis are also discussed.     

Degree of methylation of ZAC1 (PLAGL1) is associated with prenatal and post-natal growth in healthy infants of the EDEN mother child cohort

The ZAC1 gene, mapped to the 6q24 region, is part of a network of co-regulated imprinted genes involved in the control of embryonic growth. Loss of methylation at the ZAC1 differentially methylated region (DMR) is associated with transient neonatal diabetes mellitus, a developmental disorder involving growth retardation and diabetes in the first weeks of post-natal life. We assessed whether the degree of methylation of the ZAC1 DMR in leukocytes DNA extracted from cord blood is associated with fetal, birth and post-natal anthropometric measures or with C-peptide concentrations in cord serum. We also searched for an influence of dietary intake and maternal parameters on ZAC1 DMR methylation. We found positive correlations between the ZAC1 DMR methylation index (MI) and estimated fetal weight (EFW) at 32 weeks of gestation, weight at birth and weight at one year of age (respectively, r = 0.15, 0.09, 0.14; P values = 0.01, 0.15, 0.03). However, there were no significant correlations between the ZAC1 DMR MI and cord blood C-peptide levels. Maternal intakes of alcohol and of vitamins B2 were positively correlated with ZAC1 DMR methylation (respectively, r = 0.2 and 0.14; P = 0.004 and 0.04). The influence of ZAC1 seems to start in the second half of pregnancy and continue at least until the first year of life. The maternal environment also appears to contribute to the regulation of DNA methylation.     

Degree of methylation of ZAC1 (PLAGL1) is associated with prenatal and post-natal growth in healthy infants of the EDEN mother child cohort

The ZAC1 gene, mapped to the 6q24 region, is part of a network of co-regulated imprinted genes involved in the control of embryonic growth. Loss of methylation at the ZAC1 differentially methylated region (DMR) is associated with transient neonatal diabetes mellitus, a developmental disorder involving growth retardation and diabetes in the first weeks of post-natal life. We assessed whether the degree of methylation of the ZAC1 DMR in leukocytes DNA extracted from cord blood is associated with fetal, birth and post-natal anthropometric measures or with C-peptide concentrations in cord serum. We also searched for an influence of dietary intake and maternal parameters on ZAC1 DMR methylation. We found positive correlations between the ZAC1 DMR methylation index (MI) and estimated fetal weight (EFW) at 32 weeks of gestation, weight at birth and weight at one year of age (respectively, r = 0.15, 0.09, 0.14; P values = 0.01, 0.15, 0.03). However, there were no significant correlations between the ZAC1 DMR MI and cord blood C-peptide levels. Maternal intakes of alcohol and of vitamins B2 were positively correlated with ZAC1 DMR methylation (respectively, r = 0.2 and 0.14; P = 0.004 and 0.04). The influence of ZAC1 seems to start in the second half of pregnancy and continue at least until the first year of life. The maternal environment also appears to contribute to the regulation of DNA methylation.     

Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer

Background

Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.

Methodology/Principal Findings

We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.

Conclusions/Significance

These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.     

DNA Methylation Is Linked to Deacetylation of Histone H3, but Not H4, on the Imprinted Genes Snrpn and U2af1-rs1

The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpn is explored by chromatin immunoprecipitation (ChIP) and resolution of parental alleles using single-strand conformational polymorphisms. The U2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5' DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5' untranslated region (5'-UTR) and 3'-UTR of U2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins.     

Relaxation of imprinted expression of ZAC and HYMAI in a patient with transient neonatal diabetes mellitus

Transient neonatal diabetes mellitus (TNDM) is a rare disease believed to result from overexpression of a paternally expressed gene controlled by a differentially methylated CpG island on chromosome 6q24. Two genes partially overlap the island: the cell-cycle-control gene ZAC and the untranslated gene HYMAI, the function of which is currently unknown. Proof that either gene is involved in TNDM would require demonstration that imprinted expression is relaxed in TNDM patients; this has hitherto been lacking because of the rarity of the disease and the lack of imprinted expression in the lymphoblastoid cells that are generally the only resource available for study. Here, we show, for the first time, the aberrant expression of imprinted genes in a TNDM patient. In TNDM fibroblasts, the monoallelic expression of both ZAC and HYMAI is relaxed, providing strong supportive evidence that the presence of two unmethylated alleles of this locus is indeed associated with the inappropriate gene expression of neighbouring genes.     

Epigenetic status of the H19 locus in human oocytes following in vitro maturation

Imprinting is an epigenetic modification that is reprogrammed in the germ line and leads to the monoallelic expression of some genes. Imprinting involves DNA methylation. Maternal imprint is reset during oocyte growth and maturation. In vitro maturation (IVM) of oocytes may, therefore, interfere with imprint acquisition and/or maintenance. To evaluate if maturing human oocytes in vitro would be hazardous at the epigenetic level, we first determined the methylation profile of the H19 differentially methylated region (DMR). The methylation status of the H19 DMR seems particularly vulnerable to in vitro culture conditions. We analyzed oocytes at different stages of maturation following IVM, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII), using the bisulfite mutagenesis technique. Our results indicated that the unmethylated specific maternal profile for the H19 DMR was stably established at the GV stage. The majority of MI-arrested oocytes exhibited an altered pattern of methylation, the CTCF-binding site being methylated in half of the DNA strands analyzed. Of the 20 MII oocytes analyzed, 15 showed the normal unmethylated maternal pattern, while 5 originating from two different patients exhibited a methylated pattern. These findings highlight the need for extended analysis on MII-rescued oocytes to appreciate the epigenetic safety of the IVM procedure, before it becomes a routine and practical assisted reproductive procedure.     

Epimutation of the TNDM locus and the Beckwith–Wiedemann syndrome centromeric locus in individuals with transient neonatal diabetes mellitus

Transient neonatal diabetes mellitus (TNDM) is characterised by intra-uterine growth retardation, while Beckwith–Wiedemann syndrome (BWS) is a clinically heterogeneous overgrowth syndrome. Both TNDM and BWS may be caused by aberrant loss of methylation (LOM) at imprinted loci on chromosomes 6q24 and 11p15.5 respectively. Here we describe two patients with a clinical diagnosis of TNDM caused by LOM at the maternally methylated imprinted domain on 6q24; in addition, these patients had LOM at the centromeric differentially methylated region of 11p15.5. This shows that imprinting anomalies can affect more than one imprinted locus and may alter the clinical presentation of imprinted disease.     

Neonatal diabetes: new insights into aetiology and implications

Neonatal diabetes mellitus (NDM) is defined as hyperglycaemia occurring in the first few weeks of life. It can be either transient (TNDM) or permanent (PNDM), and until recently, little was known about the condition. A cohort of 30 infants with a history of TNDM has been studied, and findings have suggested that NDM does not have the same aetiology as classical type 1 childhood diabetes. Uniparental isodisomy of chromosome 6 and an unbalanced duplication of paternal chromosome 6 have both been described as a genetic basis for TNDM in over 75% of the cases. In addition, cerebellar hypoplasia and Walcott-Rallison syndrome have been associated with PNDM, suggesting an autosomal recessive inheritance pattern; furthermore, a mutation in the gene insulin promoter factor 1 has been identified as a cause of pancreatic agenesis in PNDM. In the long term, TNDM may reduce beta cell functional capacity and present a predisposition to type 2 diabetes mellitus.     

IGF2 DNA methylation is a modulator of newborn’s fetal growth and development

The insulin-like growth factor 2 (IGF2) gene, located within a cluster of imprinted genes on chromosome 11p15, encodes a fetal and placental growth factor affecting birth weight. DNA methylation variability at the IGF2 gene locus has been previously reported but its consequences on fetal growth and development are still mostly unknown in normal pediatric population. We collected one hundred placenta biopsies from 50 women with corresponding maternal and cord blood samples and measured anthropometric indices, blood pressure and metabolic phenotypes using standardized procedures. IGF2/H19 DNA methylation and IGF2 circulating levels were assessed using sodium bisulfite pyrosequencing and ELISA, respectively. Placental IGF2 (DMR0 and DMR2) DNA methylation levels were correlated with newborn’s fetal growth indices, such as weight, and with maternal IGF2 circulating concentration at the third trimester of pregnancy, whereas H19 (DMR) DNA methylation levels were correlated with IGF2 levels in cord blood. The maternal genotype of a known IGF2/H19 polymorphism (rs2107425) was associated with birth weight. Taken together, we showed that IGF2/H19 epigenotype and genotypes independently account for 31% of the newborn’s weight variance. No association was observed with maternal diabetic status, glucose concentrations or prenatal maternal body mass index. This is the first study showing that DNA methylation at the IGF2/H19 genes locus may act as a modulator of IGF2 newborn’s fetal growth and development within normal range. IGF2/H19 DNA methylation could represent a cornerstone in linking birth weight and fetal metabolic programming of late onset obesity.     

Loss of Igf2 imprinting in monoclonal mouse hepatic tumor cells is not associated with abnormal methylation patterns for the H19, Igf2, and Kvlqt1 differentially methylated regions

IGFII, the peptide encoded by the Igf2 gene, is a broad spectrum mitogen with important roles in prenatal growth as well as cancer progression. Igf2 is transcribed from the paternally inherited allele, whereas the linked H19 is transcribed from the maternal allele. Igf2 imprinting is thought to be maintained by differentially methylated regions (DMRs) located at multiple sites such as upstream of H19 and Igf2 and within Kvlqt1 loci. Biallelic expression (loss of imprinting (LOI)) of Igf2 is frequently observed in cancers, and a subset of Wilms' and intestinal tumors have been shown to exhibit abnormal methylation at H19DMR associated with loss of maternal H19 expression, but it is not known whether such changes are common in other neoplasms. Because cancers consist of diverse cell populations with and without Igf2 LOI, we established four independent monoclonal cell lines with Igf2 LOI from mouse hepatic tumors. We here demonstrate retention of normal differential methylation at H19, Igf2, or Kvlqt1 DMR by all of the cell lines. Furthermore, H19 was found to be expressed exclusively from the maternal allele, and levels of CTCF, a multifunctional nuclear factor that has an important role in the Igf2 imprinting, were comparable with those in normal hepatic tissues with no mutational changes detected. These data indicate that Igf2 LOI in tumor cells is not necessarily linked to abnormal methylation at H19, Igf2, or Kvlqt1 loci.