Congenital Imprinting Disorders Research Articles

Uncovering the phenotypic consequences of multi-locus imprinting disturbances using genome-wide methylation analysis in genomic imprinting disorders.

Imprinted genes are regulated by DNA methylation of imprinted differentially methylated regions (iDMRs). An increasing number of patients with congenital imprinting disorders (IDs) exhibit aberrant methylation at multiple imprinted loci, multi-locus imprinting disturbance (MLID). We examined MLID and its possible impact on clinical features in patients with IDs. Genome-wide DNA methylation analysis (GWMA) using blood leukocyte DNA was performed on 13 patients with Beckwith-Wiedemann syndrome (BWS), two patients with Silver-Russell syndrome (SRS), and four controls. HumanMethylation850 BeadChip analysis for 77 iDMRs (809 CpG sites) identified three patients with BWS and one patient with SRS showing additional hypomethylation, other than the disease-related iDMRs, suggestive of MLID. Two regions were aberrantly methylated in at least two patients with BWS showing MLID: PPIEL locus (chromosome 1: 39559298 to 39559744), and FAM50B locus (chromosome 6: 3849096 to 3849469). All patients with BWS- and SRS-MLID did not show any other clinical characteristics associated with additional involved iDMRs. Exome analysis in three patients with BWS who exhibited multiple hypomethylation did not identify any causative variant related to MLID. This study indicates that a genome-wide approach can unravel MLID in patients with an apparently isolated ID. Patients with MLID showed only clinical features related to the original IDs. Long-term follow-up studies in larger cohorts are warranted to evaluate any possible phenotypic consequences of other disturbed imprinted loci.

Congenital imprinting disorders

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

EP311: Diagnostic testing for Beckwith-Wiedemann syndrome/Russell-Silver syndrome: The GGC experience

Beckwith-Wiedemann syndrome (BWS) and Russell-Silver syndrome (RSS) are congenital imprinting disorders commonly caused by methylation defects in the same region of chromosome 11p15. BWS is an overgrowth syndrome that includes clinical features such as macroglossia, organomegaly, umbilical hernia, hypoglycemia, and an increased risk of childhood tumors. RSS is characterized by intrauterine and postnatal growth retardation, relative macrocephaly, fifth finger clinodactyly, triangular facies, and body asymmetry. BWS and RSS are caused by reciprocal defects of the Imprinting Control Regions (ICR) at chromosome 11p15. BWS is caused by microduplications/microdeletions of 11p15, gain of maternal methylation at ICR1, loss of maternal methylation at ICR2, paternal UPD11, and maternally inherited pathogenic variants in the CDKN1C gene. RSS is mainly caused by maternally inherited duplications of 11p15, loss of paternal methylation at ICR1, and maternal UPD7 in approximately 10% of cases. The molecular diagnostic laboratory at the Greenwood Genetic Center (GGC) offers molecular testing for BWS and RSS syndromes. Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) is the first tier of testing. This test can simultaneously detect the methylation and copy number status of the differentially methylated regions, ICR1 and ICR2, at 11p15. Abnormal MS-MLPA results are confirmed by Pyrosequencing, an orthogonal method that detects the quantitative percent of methylation at ICR1 and ICR2, which also enables the detection of mosaicism; an in-house mosaic calculator has been developed for this analysis. Cases negative for RSS by MS-MLPA can be reflexed to UPD7 testing. Sanger sequencing of the CDKN1C gene and UPD7 testing are available at GGC as reflex tests for cases with negative BWS MLPA or RSS MLPA results, respectively. A total of 867 cases have been included in this study. For BWS, the diagnostic yield by MS-MLPA is 21.6% (117/541), where loss of methylation at ICR2 (87 cases), gain of methylation at ICR1 (3 cases), and copy number change (5 cases) were detected. Among these BWS MLPA positive cases, 18.8% (22/117) were due to paternal UPD11, which included six cases that resulted from mosaicism. Approximately, 23.3% (99/424) of BWS MLPA-negative cases had CDKN1C sequencing, and variants of uncertain clinical significance (VUS) were identified in four cases. For RSS, the diagnostic yield by MS-MLPA is 12.5% (41/326) of which three cases had a mosaic loss of methylation at ICR1 and two cases had copy number changes. 21.7% (62/285) of RSS MLPA-negative cases were reflexed to UPD7 testing, which increased the diagnostic yield to 14% (46/326). It is of interest to note that eight cases submitted for BWS testing were found to be abnormal for RSS syndrome. The clinical finding of hemihypertrophy in these cases prompted testing for BWS; this raises the question of whether hemihypoplasia was being interpreted as hemihypertrophy in these cases since loss of methylation at ICR1 has been observed in isolated cases of hemihypoplasia. Similarly, two cases that were submitted for RSS testing was found to be abnormal for BWS. This finding indicates that hypomethylation of both ICR1 and ICR2 has been identified in patients with RSS, and it is thought that epigenetic mosaicism may exist between relevant disease tissues, which can result in clinical expression of either RSS or BWS. In summary, by utilizing various methods of testing for BWS/RSS (MS-MLPA, Pyrosequencing, Sanger sequencing, and UPD testing), the collective diagnostic yield is approximately 19%. Pyrosequencing can confirm an abnormal finding by MS-MLPA and is a quantitative method that can be utilized for detecting percent mosaicism. Finally, for RSS negative cases, reflex testing for UPD7 can increase the diagnostic yield.

ImprintSeq, a novel tool to interrogate DNA methylation at human imprinted regions and diagnose multilocus imprinting disturbance

PurposeDisruptions of genomic imprinting are associated with congenital imprinting disorders (CIDs) and other disease states, including cancer. CIDs are most often associated with altered methylation at imprinted differentially methylated regions (iDMRs). In some cases, multiple iDMRs are affected causing multilocus imprinting disturbances (MLIDs). The availability of accurate, quantitative, and scalable high-throughput methods to interrogate multiple iDMRs simultaneously would enhance clinical diagnostics and research. MethodsWe report the development of a custom targeted methylation sequencing panel that covered most relevant 63 iDMRs for CIDs and the detection of MLIDs. We tested it in 70 healthy controls and 147 individuals with CIDs. We distinguished loss and gain of methylation per differentially methylated region and classified high and moderate methylation alterations. ResultsAcross a range of CIDs with a variety of molecular mechanisms, ImprintSeq performed at 98.4% sensitivity, 99.9% specificity, and 99.9% accuracy (when compared with previous diagnostic testing). ImprintSeq was highly sensitive for detecting MLIDs and enabled diagnostic criteria for MLID to be proposed. In a child with extreme MLID profile a probable genetic cause was identified. ConclusionImprintSeq provides a novel assay for clinical diagnostic and research studies of CIDs, MLIDs, and the role of disordered imprinting in human disease states.

Alteration of Genomic Imprinting after Assisted Reproductive Technologies and Long-Term Health.

Assisted reproductive technologies (ART) are the treatment of choice for some infertile couples and even though these procedures are generally considered safe, children conceived by ART have shown higher reported risks of some perinatal and postnatal complications such as low birth weight, preterm birth, and childhood cancer. In addition, the frequency of some congenital imprinting disorders, like Beckwith–Wiedemann Syndrome and Silver–Russell Syndrome, is higher than expected in the general population after ART. Experimental evidence from animal studies suggests that ART can induce stress in the embryo and influence gene expression and DNA methylation. Human epigenome studies have generally revealed an enrichment of alterations in imprinted regions in children conceived by ART, but no global methylation alterations. ART procedures occur simultaneously with the establishment and maintenance of imprinting during embryonic development, so this may underlie the apparent sensitivity of imprinted regions to ART. The impact in adulthood of imprinting alterations that occurred during early embryonic development is still unclear, but some experimental evidence in mice showed higher risk to obesity and cardiovascular disease after the restriction of some imprinted genes in early embryonic development. This supports the hypothesis that imprinting alterations in early development might induce epigenetic programming of metabolism and affect long-term health. Given the growing use of ART, it is important to determine the impact of ART in genomic imprinting and long-term health.

Basics and disturbances of genomic imprinting

Abstract Genomic imprinting ensures the parent-specific expression of either the maternal or the paternal allele, by different epigenetic processes (DNA methylation and histone modifications) that confer parent-specific marks (imprints) in the paternal and maternal germline, respectively. Most protein-coding imprinted genes are involved in embryonic growth, development, and behavior. They are usually organized in genomic domains that are regulated by differentially methylated regions (DMRs). Genomic imprints are erased in the primordial germ cells and then reset in a gene-specific manner according to the sex of the germline. The imprinted genes regulate and interact with other genes, consistent with the existence of an imprinted gene network. Defects of genomic imprinting result in syndromal imprinting disorders. To date a dozen congenital imprinting disorders are known. Usually, a given imprinting disorder can be caused by different types of defects, including point mutations, deletions/duplications, uniparental disomy, and epimutations. Causative trans-acting factors in imprinting disorders, including ZFP57 and the subcortical maternal complex (SCMC), have the potential to affect multiple DMRs across the genome, resulting in a multi-locus imprinting disturbance. There is evidence that mutations in components of the SCMC can confer an increased risk for imprinting disorders.

Imprinting disorders: a group of congenital disorders with overlapping patterns of molecular changes affecting imprinted loci.

Congenital imprinting disorders (IDs) are characterised by molecular changes affecting imprinted chromosomal regions and genes, i.e. genes that are expressed in a parent-of-origin specific manner. Recent years have seen a great expansion in the range of alterations in regulation, dosage or DNA sequence shown to disturb imprinted gene expression, and the correspondingly broad range of resultant clinical syndromes. At the same time, however, it has become clear that this diversity of IDs has common underlying principles, not only in shared molecular mechanisms, but also in interrelated clinical impacts upon growth, development and metabolism. Thus, detailed and systematic analysis of IDs can not only identify unifying principles of molecular epigenetics in health and disease, but also support personalisation of diagnosis and management for individual patients and families.

Congenital imprinting disorders: EUCID.net - a network to decipher their aetiology and to improve the diagnostic and clinical care.

Imprinting disorders (IDs) are a group of eight rare but probably underdiagnosed congenital diseases affecting growth, development and metabolism. They are caused by similar molecular changes affecting regulation, dosage or the genomic sequence of imprinted genes. Each ID is characterised by specific clinical features, and, as each appeared to be associated with specific imprinting defects, they have been widely regarded as separate entities. However, they share clinical characteristics and can show overlapping molecular alterations. Nevertheless, IDs are usually studied separately despite their common underlying (epi)genetic aetiologies, and their basic pathogenesis and long-term clinical consequences remain largely unknown. Efforts to elucidate the aetiology of IDs are currently fragmented across Europe, and standardisation of diagnostic and clinical management is lacking. The new consortium EUCID.net (European network of congenital imprinting disorders) now aims to promote better clinical care and scientific investigation of imprinting disorders by establishing a concerted multidisciplinary alliance of clinicians, researchers, patients and families. By encompassing all IDs and establishing a wide ranging and collaborative network, EUCID.net brings together a wide variety of expertise and interests to engender new collaborations and initiatives.

Multilocus methylation defects in imprinting disorders.

Mammals inherit two complete sets of chromosomes, one from the father and one from the mother, and most autosomal genes are expressed from both maternal and paternal alleles. In imprinted genes, the expression of the allele is dependent upon its parental origin. Appropriate regulation of imprinted genes is important for normal development, with several genetic diseases associated with imprinting defects. A common process for controlling gene activity is methylation. The first steps for understanding the functions of DNA methylation and its regulation in mammalian development have led us to identify common (epi)genetic mechanisms involved in the eight human congenital imprinting disorders.

Epigenetic Deregulation of Genomic Imprinting in Humans: Causal Mechanisms and Clinical Implications

Mammalian genes controlled by genomic imprinting play important roles in development and diverse postnatal processes. A growing number of congenital disorders have been linked to genomic imprinting. Each of these is caused by perturbed gene expression at one principal imprinted domain. Some imprinting disorders, including the Prader-Willi and Angelman syndromes, are caused almost exclusively by genetic mutations. In several others, including the Beckwith-Wiedemann and Silver-Russell growth syndromes, and transient neonatal diabetes mellitus, imprinted expression is perturbed mostly by epigenetic alterations at 'imprinting control regions' and at other specific regulatory sequences. In a minority of these patients, DNA methylation is altered at multiple imprinted loci, suggesting that common trans-acting factors are affected. Here, we review the epimutations involved in congenital imprinting disorders and the associated clinical features. Trans-acting factors known to be causally involved are discussed and other trans-acting factors that are potentially implicated are also presented.

Congenital Imprinting Disorders: A Novel Mechanism Linking Seemingly Unrelated Disorders

Congenital Imprinting Disorders: A Novel Mechanism Linking Seemingly Unrelated Disorders

Use of multilocus methylation-specific single nucleotide primer extension (MS-SNuPE) technology in diagnostic testing for human imprinted loci

A number of diseases have been found to be linked to aberrant methylation of specific genes. However, most of the routine diagnostic techniques to detect epigenetic disturbances are restricted to single loci. Additionally, a precise quantification of the methylation status is often hampered. A considerable fraction of patients with Silver-Russell syndrome, Beckwith-Wiedemann syndrome and transient neonatal diabetes mellitus exhibit loss of methylation at further imprinted loci in addition to the disease specific ones (multilocus methylation defects, MLMD). As the currently available tests are mainly focused on single imprinted loci on different chromosomes and thereby make the detection of multilocus methylation defects time-consuming and expensive, we established methylation-specific single nucleotide primer extension (MS-SNuPE) assays for a simultaneous quantification of methylation at multiple methylated loci. We chose loci generally affected in patients with MLMD. The method was validated by screening 66 individuals with known (epi)genetic disturbances. In comparison to other methylation-specific techniques, multilocus methylation-specific single nucleotide primer extension allows the quantitative analysis of numerous CpG islands of different loci in one assay and is, therefore, suitable for the simultaneous diagnostic testing for different congenital imprinting disorders in parallel, as well as for MLMD.

Segmental maternal uniparental disomy 7q associated with DLK1/GTL2 (14q32) hypomethylation

Aberrant methylation at different imprinted loci has been reported for several congenital imprinting disorders, that is, Silver-Russell syndrome (SRS), but the coincidental occurrence of aberrant methylation and uniparental disomy (UPD) has not yet been described. We report on a patient initially diagnosed with SRS carrying a segmental maternal UPD of chromosome 7 [upd(7q)mat]. By further screening the patient's DNA for methylation defects on other chromosomes we identified a hypomethylation of the paternally methylated DLK1/GTL2 locus in 14q32, an epigenotype typically associated with the upd(14)mat phenotype. Detailed clinical analysis confirmed the molecular finding in the patient indicating that the 14q32 epimutation was clinically preponderant. The parallel occurrence of upd(7q)mat and a DLK1/GTL2 hypomethylation in the same patient is a unique finding. Indeed, both disturbances might have occurred coincidentally, but it can also be hypothesized that the upd(7q)mat as the initial genomic mutation represents a trans-acting mutation causing an aberrant methylation in 14q32.

Silver-Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues

In all known congenital imprinting disorders an association with aberrant methylation or mutations at specific loci was well established. However, several patients with transient neonatal diabetes mellitus (TNDM), Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) exhibiting multilocus hypomethylation (MLH) have meanwhile been described. Whereas TNDM patients with MLH show clinical symptoms different from carriers with isolated 6q24 aberrations, MLH carriers diagnosed as BWS or SRS present only the syndrome-specific features. Interestingly, SRS and BWS patients with nearly identical MLH patterns in leukocytes have been identified. We now report on the molecular findings in DNA in three SRS patients with hypomethylation of both 11p15 imprinted control regions (ICRs) in leukocytes. One patient was a monozygotic (MZ) twin, another was a triplet. While the hypomethylation affected both oppositely imprinted 11p15 ICRs in leukocytes, in buccal swab DNA only the ICR1 hypomethylation was visible in two of our patients. In the non-affected MZ twin of one of these patients, aberrant methylation was also present in leukocytes but neither in buccal swab DNA nor in skin fibroblasts. Despite mutation screening of several factors involved in establishment and maintenance of methylation marks including ZFP57, MBD3, DNMT1 and DNMT3L the molecular clue for the ICR1/ICR2 hypomethylation in our patients remained unclear. Furthermore, the reason for the development of the specific SRS phenotype is not obvious. In conclusion, our data reflect the broad range of epimutations in SRS and illustrate that an extensive molecular and clinical characterization of patients is necessary.

Silver-Russell syndrome: genetic basis and molecular genetic testing

Imprinted genes with a parent-of-origin specific expression are involved in various aspects of growth that are rooted in the prenatal period. Therefore it is predictable that many of the so far known congenital imprinting disorders (IDs) are clinically characterised by growth disturbances. A noteable imprinting disorder is Silver-Russell syndrome (SRS), a congenital disease characterised by intrauterine and postnatal growth retardation, relative macrocephaly, a typical triangular face, asymmetry and further less characteristic features. However, the clinical spectrum is broad and the clinical diagnosis often subjective. Genetic and epigenetic disturbances can meanwhile be detected in approximately 50% of patients with typical SRS features. Nearly one tenth of patients carry a maternal uniparental disomy of chromosome 7 (UPD(7)mat), more than 38% show a hypomethylation in the imprinting control region 1 in 11p15. More than 1% of patients show (sub)microscopic chromosomal aberrations. Interestingly, in ~7% of 11p15 hypomethylation carriers, demethylation of other imprinted loci can be detected. Clinically, these patients do not differ from those with isolated 11p15 hypomethylation whereas the UPD(7)mat patients generally show a milder phenotype. However, an unambiguous (epi)genotype-phenotype correlation can not be delineated.We therefore suggest a diagnostic algorithm focused on the 11p15 hypomethylation, UPD(7)mat and cryptic chromosomal imbalances for patients with typical SRS phenotype, but also with milder clinical signs only reminiscent for the disease.