Hypomethylation of a centromeric block of ICR1 is sufficient to cause Silver-Russell syndrome

Silver-Russell syndrome (SRS) is a representative imprinting disorder. A major cause is the loss of methylation (LOM) of imprinting control region 1 (ICR1) within the IGF2/H19 domain. ICR1 is a gametic differentially methylated region (DMR) consisting of two repeat blocks, with each block including three CTCF target sites (CTSs). ICR1-LOM on the paternal allele allows CTCF to bind to CTSs, resulting in IGF2 repression on the paternal allele and biallelic expression of H19. We analysed 10 differentially methylated sites (DMSs) (ie, seven CTSs and three somatic DMRs within the IGF2/H19 domain, including two IGF2-DMRs and the H19-promoter) in five SRS patients with ICR1-LOM. Four patients showed consistent hypomethylation at all DMSs; however, one exhibited a peculiar LOM pattern, showing LOM at the centromeric region of the IGF2/H19 domain but normal methylation at the telomeric region. This raised important points: there may be a separate regulation of DNA methylation for the two repeat blocks within ICR1; there is independent control of somatic DMRs under each repeat block; sufficient IGF2 repression to cause SRS phenotypes occurs by LOM only in the centromeric block; and the need for simultaneous methylation analysis of several DMSs in both blocks for a correct molecular diagnosis.

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An upstream insulator regulates DLK1 imprinting in AML

Blood ◽

10.1182/blood-2009-03-212746 ◽

2010 ◽

Vol 115 (11) ◽

pp. 2260-2263 ◽

Cited By ~ 34

Author(s):

Haytham Khoury ◽

Fernando Suarez-Saiz ◽

Samantha Wu ◽

Mark D. Minden

Keyword(s):

Bone Marrow ◽

Strong Association ◽

Differentially Methylated Region ◽

Normal Bone Marrow ◽

Nucleotide Polymorphisms ◽

Methylation Analysis ◽

Biallelic Expression ◽

Normal Bone ◽

Allele Specific ◽

Allele Specific Methylation

Abstract DLK1 is an imprinted gene on chromosome 14. Using informative coding single nucleotide polymorphisms, we found DLK1 expression to be monoallelic in normal bone marrow, whereas it was biallelic in 76% of acute myeloid leukemia (AML) overexpressing DLK1 (61% of all AML). Quantitative methylation analysis of 7 cytosine-phosphate-guanosine-rich areas (3 upstream of or within DLK1, the putative intergenic-differentially methylated region and 3 upstream of or within MEG3) revealed a strong association between biallelic DLK1 expression and hypermethylation of a cytosine-phosphate-guanosine-rich region 18 kb upstream of DLK1. Allele-specific methylation analysis of this region revealed the alleles to be differentially methylated in normal bone marrow and monoallelic DLK1 AML, whereas there was increased methylation of both alleles in AML with biallelic expression. Moreover, chromatin immunoprecipitation analysis revealed that CCTC-binding factor binds to this region in monoallelic but not biallelic expression samples. Taken together, our data indicate that an insulator located 18 kb upstream of DLK1 plays an important role in regulating DLK1 imprinting.

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Somatotroph Tumors and the Epigenetic Status of the GNAS Locus

International Journal of Molecular Sciences ◽

10.3390/ijms22147570 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7570

Author(s):

Pauline Romanet ◽

Justine Galluso ◽

Peter Kamenicky ◽

Mirella Hage ◽

Marily Theodoropoulou ◽

...

Keyword(s):

Genomic Analysis ◽

Somatostatin Analogues ◽

Differentially Methylated Region ◽

Monoallelic Expression ◽

Lower Sensitivity ◽

Biallelic Expression ◽

Expression Levels ◽

Methylation Index ◽

Significant Difference ◽

Gsp Oncogene

Background: Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp-negative somatotroph tumors, GNAS expression itself is highly variable; those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp-negative tumors affect GNAS expression levels and tumorigenesis. Methods: We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Results: Multi-omics analysis of the first cohort identified a significant difference between gsp-negative and gsp-positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp-negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Conclusion: Altered A/B DMR methylation was found exclusively in gsp-negative somatotroph tumors. 43% of gsp-negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS, SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.

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Analysis of the Functions of Recombination-Related Genes in the Generation of Large Chromosomal Deletions by Loop-Out Recombination in Aspergillus oryzae

Eukaryotic Cell ◽

10.1128/ec.05208-11 ◽

2012 ◽

Vol 11 (4) ◽

pp. 507-517 ◽

Cited By ~ 7

Author(s):

Tadashi Takahashi ◽

Masahiro Ogawa ◽

Yasuji Koyama

Keyword(s):

Aspergillus Oryzae ◽

Chromosomal Region ◽

Centromeric Region ◽

Chromosome 8 ◽

Telomeric Region ◽

Dna Double Strand Breaks ◽

Chromosomal Structure ◽

Content Type ◽

Strand Breaks ◽

Chromosomal Deletions

ABSTRACT Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70 , ligD , rad52 , rad54 , and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae . The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δ ku70 and Δ ku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the Δ ligD , Δ ku70-rad52 , and Δ ku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δ ku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD , rad52 , and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure.

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Mosaic Segmental and Whole-Chromosome upd(11)mat in Silver-Russell Syndrome

Genes ◽

10.3390/genes12040581 ◽

2021 ◽

Vol 12 (4) ◽

pp. 581

Author(s):

Laura Pignata ◽

Angela Sparago ◽

Orazio Palumbo ◽

Elena Andreucci ◽

Elisabetta Lapi ◽

...

Keyword(s):

Molecular Level ◽

Opposite Sign ◽

Uniparental Disomy ◽

Differentially Methylated Region ◽

Imprinted Genes ◽

Loss Of Function ◽

Growth Disturbances ◽

Molecular Defects ◽

Entire Chromosome ◽

Silver Russell Syndrome

Molecular defects altering the expression of the imprinted genes of the 11p15.5 cluster are responsible for the etiology of two congenital disorders characterized by opposite growth disturbances, Silver–Russell syndrome (SRS), associated with growth restriction, and Beckwith–Wiedemann syndrome (BWS), associated with overgrowth. At the molecular level, SRS and BWS are characterized by defects of opposite sign, including loss (LoM) or gain (GoM) of methylation at the H19/IGF2:intergenic differentially methylated region (H19/IGF2:IG-DMR), maternal or paternal duplication (dup) of 11p15.5, maternal (mat) or paternal (pat) uniparental disomy (upd), and gain or loss of function mutations of CDKN1C. However, while upd(11)pat is found in 20% of BWS cases and in the majority of them it is segmental, upd(11)mat is extremely rare, being reported in only two SRS cases to date, and in both of them is extended to the whole chromosome. Here, we report on two novel cases of mosaic upd(11)mat with SRS phenotype. The upd is mosaic and isodisomic in both cases but covers the entire chromosome in one case and is restricted to 11p14.1-pter in the other case. The segmental upd(11)mat adds further to the list of molecular defects of opposite sign in SRS and BWS, making these two imprinting disorders even more specular than previously described.

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Novel conserved elements upstream of theH19gene are transcribed and act as mesodermal enhancers

Development ◽

10.1242/dev.129.5.1205 ◽

2002 ◽

Vol 129 (5) ◽

pp. 1205-1213

Author(s):

Robert A. Drewell ◽

Katharine L. Arney ◽

Takahiro Arima ◽

Sheila C. Barton ◽

James D. Brenton ◽

...

Keyword(s):

Gene Expression ◽

Expression Pattern ◽

Reporter Gene ◽

Differentially Methylated Region ◽

Genomic Sequencing ◽

Strong Homology ◽

Imprinting Control Region ◽

Enhancer Function ◽

Human And Mouse ◽

Mouse Genomic

The reciprocally imprinted H19 and Igf2 genes form a co-ordinately regulated 130 kb unit in the mouse controlled by widely dispersed enhancers, epigenetically modified silencers and an imprinting control region (ICR). Comparative human and mouse genomic sequencing between H19 and Igf2 revealed two novel regions of strong homology upstream of the ICR termed H19 upstream conserved regions (HUCs). Mouse HUC1 and HUC2 act as potent enhancers capable of driving expression of an H19 reporter gene in a range of mesodermal tissues. Intriguingly, the HUC sequences are also transcribed bi-allelically in mouse and human, but their expression pattern in neural and endodermal tissues in day 13.5 embryos is distinct from their enhancer function. The location of the HUC mesodermal enhancers upstream of the ICR and H19, and their capacity for interaction with both H19 and Igf2 requires critical re-evaluation of the cis-regulation of imprinted gene expression of H19 and Igf2 in a range of mesodermal tissues. We propose that these novel sequences interact with the ICR at H19 and the epigenetically regulated silencer at differentially methylated region 1 (DMR1) of Igf2.

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Allele-specific in situ hybridization (ASISH) analysis: a novel technique which resolves differential allelic usage of H19 within the same cell lineage during human placental development

Development ◽

10.1242/dev.122.3.839 ◽

1996 ◽

Vol 122 (3) ◽

pp. 839-847 ◽

Cited By ~ 1

Author(s):

G.I. Adam ◽

H. Cui ◽

S.J. Miller ◽

F. Flam ◽

R. Ohlsson

Keyword(s):

In Situ Hybridization ◽

First Trimester ◽

Paternal Allele ◽

Monoallelic Expression ◽

Hybridization Technique ◽

Placental Development ◽

Foetal Development ◽

Biallelic Expression ◽

Normal Human

Precursory studies of H19 transcription during human foetal development have demonstrated maternally derived monoallelic expression. Analyses in extra-embryonic tissues, however, have been more equivocal, with discernible levels of expression of the paternal allele of H19 documented in the first trimester placenta. By refining the in situ hybridization technique we have developed an assay to enable the functional imprinting status of H19 to be determined at the cellular level. This assay involves the use of oligonucleotide DNA probes that are able to discriminate between allelic RNA transcripts containing sequence polymorphisms. Biallelic expression of H19 is confined to a subpopulation of cells of the trophoblast lineage, the extravillous cytotrophoblast, while the mesenchymal stroma cells maintain the imprinted pattern of monoallelic expression of H19 throughout placental development. This data demonstrates that the low level of paternal H19 expression previously detected in normal human placenta is not due to a random loss of functional imprinting, but appears to result from a developmentally regulated cell type-specific activation of the paternal allele. In addition, biallelic expression of H19 does not seem to affect the functional imprinting of the insulin-like growth factor II gene, which is monoallelically expressed at relatively high levels in the extra-villous cytotrophoblasts. These results imply that the allelic usage of these two genes in normal human placental development may not be directly analogous to the situation previously documented in the mouse embryo.

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Severe in utero under-virilization in a 46,XY patient with Silver-Russell syndrome with 11p15 loss of methylation

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2018-0464 ◽

2019 ◽

Vol 32 (2) ◽

pp. 191-196

Author(s):

Masanori Adachi ◽

Maki Fukami ◽

Masayo Kagami ◽

Noriko Sho ◽

Yuichiro Yamazaki ◽

...

Keyword(s):

Failure To Thrive ◽

Differentially Methylated Region ◽

Undescended Testes ◽

Case Presentation ◽

Body Asymmetry ◽

Sex Development ◽

Pathogenic Variants ◽

Hormone Analog ◽

Differences Of Sex Development ◽

Silver Russell Syndrome

Abstract Background Silver-Russell syndrome (SRS) is characterized by growth retardation and variable features including macrocephaly, body asymmetry, and genital manifestations such as cryptorchidism in 46,XY patients. Case presentation The patient was born at 39 weeks with a birth weight of 1344 g. Subtle clitoromegaly warranted a thorough evaluation, which disclosed 46,XY karyotype, bilateral undescended testes, and a rudimentary uterus. Because of severe under-virilization, the patient was assigned as female. Failure to thrive, macrocephaly, and body asymmetry led to the diagnosis of SRS, confirmed by marked hypomethylation of H19/IGF2 intergenic differentially methylated region (IG-DMR). From age 9 years, progressive virilization occurred, which necessitated luteinizing hormone-releasing hormone analog (LHRHa) treatment. Gonadal resection at 15 years revealed immature testes with mostly Sertoli-cell-only tubules. Panel analysis for 46,XY-differences of sex development (DSD) failed to detect any pathogenic variants. Conclusions This is the second reported case of molecularly proven 46,XY SRS accompanied by severe under-virilization. SRS should be included in the differential diagnosis of 46,XY-DSD.

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Multiple Nucleosome Positioning Sites Regulate the CTCF-Mediated Insulator Function of the H19 Imprinting Control Region†

Molecular and Cellular Biology ◽

10.1128/mcb.22.10.3339-3344.2002 ◽

2002 ◽

Vol 22 (10) ◽

pp. 3339-3344 ◽

Cited By ~ 37

Author(s):

Meena Kanduri ◽

Chandrasekhar Kanduri ◽

Piero Mariano ◽

Alexander A. Vostrov ◽

Wolfgang Quitschke ◽

...

Keyword(s):

Control Region ◽

Insertional Mutagenesis ◽

Site Occupancy ◽

Nucleosome Positioning ◽

Target Site ◽

Chromatin Insulator ◽

Insulator Function ◽

Target Sites ◽

Imprinting Control Region ◽

Precise Distribution

ABSTRACT The 5′ region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.

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The Mouse Murr1 Gene Is Imprinted in the Adult Brain, Presumably Due to Transcriptional Interference by the Antisense-Oriented U2af1-rs1 Gene

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.270-279.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 270-279 ◽

Cited By ~ 59

Author(s):

Youdong Wang ◽

Keiichiro Joh ◽

Sadahiko Masuko ◽

Hitomi Yatsuki ◽

Hidenobu Soejima ◽

...

Keyword(s):

Developmental Change ◽

Adult Brain ◽

Paternal Allele ◽

Transcriptional Interference ◽

Specific Expression ◽

Biallelic Expression ◽

Allele Specific ◽

The Brain ◽

Predominant Expression

ABSTRACT The mouse Murr1 gene contains an imprinted gene, U2af1-rs1, in its first intron. U2af1-rs1 shows paternal allele-specific expression and is transcribed in the direction opposite to that of the Murr1 gene. In contrast to a previous report of biallelic expression of Murr1 in neonatal mice, we have found that the maternal allele is expressed predominantly in the adult brain and also preferentially in other adult tissues. This maternal-predominant expression is not observed in embryonic and neonatal brains. In situ hybridization experiments that used the adult brain indicated that Murr1 gene was maternally expressed in neuronal cells in all regions of the brain. We analyzed the developmental change in the expression levels of both Murr1 and U2af1-rs1 in the brain and liver, and we propose that the maternal-predominant expression of Murr1 results from transcriptional interference of the gene by U2af1-rs1 through the Murr1 promoter region.

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