Humanized H19/Igf2 locus reveals diverged imprinting mechanism between mouse and human and reflects Silver–Russell syndrome phenotypes

Genomic imprinting is essential for mammalian development. Curiously, elements that regulate genomic imprinting, the imprinting control regions (ICRs), often diverge across species. To understand whether the diverged ICR sequence plays a species-specific role at the H19/insulin-like growth factor 2 (Igf2) imprinted locus, we generated a mouse in which the human ICR (hIC1) sequence replaced the endogenous mouse ICR. We show that the imprinting mechanism has partially diverged between mouse and human, depending on the parental origin of the hIC1 in mouse. We also suggest that our mouse model is optimal for studying the imprinting disorders Beckwith–Wiedemann syndrome when hIC1 is maternally transmitted, and Silver–Russell syndrome when hIC1 is paternally transmitted

Co-occurrence of Beckwith-Wiedemann syndrome and pseudohypoparathyroidism type 1B: coincidence or common molecular mechanism?

Imprinting disorders are congenital diseases caused by dysregulation of genomic imprinting, affecting growth, neurocognitive development, metabolism and cancer predisposition. Overlapping clinical features are often observed among this group of diseases. In rare cases, two fully expressed imprinting disorders may coexist in the same patient. A dozen cases of this type have been reported so far. Most of them are represented by individuals affected by Beckwith–Wiedemann spectrum (BWSp) and Transient Neonatal Diabetes Mellitus (TNDM) or BWSp and Pseudo-hypoparathyroidism type 1B (PHP1B). All these patients displayed Multilocus imprinting disturbances (MLID). Here, we report the first case of co-occurrence of BWS and PHP1B in the same individual in absence of MLID. Genome-wide methylation and SNP-array analyses demonstrated loss of methylation of the KCNQ1OT1:TSS-DMR on chromosome 11p15.5 as molecular cause of BWSp, and upd(20)pat as cause of PHP1B. The absence of MLID and the heterodisomy of chromosome 20 suggests that BWSp and PHP1B arose through distinct and independent mechanism in our patient. However, we cannot exclude that the rare combination of the epigenetic defect on chromosome 11 and the UPD on chromosome 20 may originate from a common so far undetermined predisposing molecular lesion. A better comprehension of the molecular mechanisms underlying the co-occurrence of two imprinting disorders will improve genetic counselling and estimate of familial recurrence risk of these rare cases. Furthermore, our study also supports the importance of multilocus molecular testing for revealing MLID as well as complex cases of imprinting disorders

11.Hypoxic ventilatory depressionと思われる一症例について(第551回千葉医学会例会・第9回麻酔科例会・第18回千葉麻酔懇話会)

FISH analysis on metaphase nuclei (top panel) of cultured cells derived from peripheral blood leukocytes of the proband of family 2 by using BAC probes for 11p15.5-15.4 (RP11-11A9, 3,236,552-3,356,012, green) and 11q22.3 (RP11-179B7, 104,298,339-104,459,797, red). The green signal on both homologues is visible only at chr11p, demonstrating the presence of an in cis duplication and excluding an unbalanced translocation. FISH analysis on interphase nuclei (bottom panel) using the BACs RP11-699D10 (2.9–3.0 Mb, red) and RP11-11A9 (green), hybridizing within the duplication. Note that single and duplicated signals can be seen on the two homologues, respectively. The red-green-green-red order of the duplicated signals indicates that the duplication is inverted. (PDF 52 kb

Clinical and molecular characterization of patients affected by Beckwith-Wiedemann spectrum conceived through assisted reproduction techniques

The prevalence of Beckwith-Wiedemann spectrum (BWSp) is tenfold increased in children conceived through assisted reproductive techniques (ART). More than 90% of ART-BWSp patients reported so far display imprinting center 2 loss-of-methylations (IC2-LoM), versus 50% of naturally conceived BWSp patients. We describe a cohort of 74 ART-BWSp patients comparing their features with a cohort of naturally conceived BWSp patients, with the ART-BWSp patients previously described in literature, and with the general population of children born from ART. We found that the distribution of UPD(11)pat was not significantly different in ART and naturally conceived patients. We observed 68.9% of IC2-LoM and 16.2% of mosaic UPD(11)pat in our ART cohort, that strongly differ from the figure reported in other cohorts so far. Since UPD(11)pat likely results from post-fertilization recombination events, our findings allows to hypothesize that more complex molecular mechanisms, besides methylation disturbances, may underlie BWSp increased risk in ART pregnancies. Moreover, comparing the clinical features of ART and non-ART BWSp patients, we found that ART-BWSp patients might have a milder phenotype. Finally, our data show a progressive increase in the prevalence of BWSp over time, paralleling that of ART usage in the last decades

The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derived from the study of Beckwith–Wiedemann syndrome and Silver–Russell syndrome cases

A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver–Russell syndrome (SRS) and Beckwith–Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5′ 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA

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 IC

Is ZFP57 binding to H19/IGF2: IG-DMR affected in Silver-Russell syndrome?

BACKGROUND: Loss of paternal methylation (LOM) of the H19/IGF2 intergenic differentially methylated region (H19/IGF2:IG-DMR) causes alteration of H19/IGF2 imprinting and Silver-Russell syndrome (SRS). Recently, internal deletions of the H19/IGF2:IG-DMR have been associated with LOM and SRS when present on the paternal chromosome. In contrast, previously described deletions, most of which cause gain of methylation (GOM) and Beckwith-Wiedemann syndrome (BWS) on maternal transmission, were consistently associated with normal methylation and phenotype if paternally inherited. PRESENTATION OF THE HYPOTHESIS: The presence of several target sites (ZTSs) and three demonstrated binding regions (BRs) for the imprinting factor ZFP57 in the H19/IGF2:IG-DMR suggest the involvement of this factor in the maintenance of methylation of this locus. By comparing the extension of the H19/IGF2:IG-DMR deletions with the binding profile of ZFP57, we propose that the effect of the deletions on DNA methylation and clinical phenotype is dependent on their interference with ZFP57 binding. Indeed, deletions strongly affecting a ZFP57 BR result in LOM and SRS, while deletions preserving a significant number of ZFPs in each BR do not alter methylation and are associated with normal phenotype. TESTING THE HYPOTHESIS: The generation of transgenic mouse lines in which the endogenous H19/IGF2:IG-DMR is replaced by the human orthologous locus including the three ZFP57 BRs or their mutant versions will allow to test the role of ZFP57 binding in imprinted methylation and growth phenotype. IMPLICATIONS OF THE HYPOTHESIS: Similarly to what is proposed for maternally inherited BWS mutations and CTCF and OCT4/SOX2 binding, we suggest that deletions of the H19/IGF2:IG-DMR result in SRS with LOM if ZFP57 binding on the paternal chromosome is affected.Background: Loss of paternal methylation (LOM) of the H19/IGF2 intergenic differentially methylated region (H19/IGF2: IG-DMR) causes alteration of H19/IGF2 imprinting and Silver-Russell syndrome (SRS). Recently, internal deletions of the H19/IGF2: IG-DMR have been associated with LOM and SRS when present on the paternal chromosome. In contrast, previously described deletions, most of which cause gain of methylation (GOM) and Beckwith-Wiedemann syndrome (BWS) on maternal transmission, were consistently associated with normal methylation and phenotype if paternally inherited.Presentation of the hypothesis: The presence of several target sites (ZTSs) and three demonstrated binding regions (BRs) for the imprinting factor ZFP57 in the H19/IGF2: IG-DMR suggest the involvement of this factor in the maintenance of methylation of this locus. By comparing the extension of the H19/IGF2: IG-DMR deletions with the binding profile of ZFP57, we propose that the effect of the deletions on DNA methylation and clinical phenotype is dependent on their interference with ZFP57 binding. Indeed, deletions strongly affecting a ZFP57 BR result in LOM and SRS, while deletions preserving a significant number of ZFPs in each BR do not alter methylation and are associated with normal phenotype.Testing the hypothesis: The generation of transgenic mouse lines in which the endogenous H19/IGF2: IG-DMR is replaced by the human orthologous locus including the three ZFP57 BRs or their mutant versions will allow to test the role of ZFP57 binding in imprinted methylation and growth phenotype.Implications of the hypothesis: Similarly to what is proposed for maternally inherited BWS mutations and CTCF and OCT4/SOX2 binding, we suggest that deletions of the H19/IGF2: IG-DMR result in SRS with LOM if ZFP57 binding on the paternal chromosome is affected

Additional file 1: of Is ZFP57 binding to H19/IGF2:IG-DMR affected in Silver-Russell syndrome?

Table S1. List of CTCF, OCT4/SOX2 and ZFP57 binding regions and potential target sites in the H19/IGF2:IG-DMR. Genomic positions of the binding regions (if demonstrated) and potential target sites and motifs of all these factors have been listed. (PDF 86 kb

Current distribution effects in organic sexithiophene FETs investigated by lock-in thermography: mobility evaluation issues

In this letter, a lock-in thermography technique has been used to investigate the actual current distribution profile in the active channel region of organic FETs (oFETs). The high accuracy of the setup, for the first time, shows an evidence of not uniformity in the current flow over the device area. The physical origin of this experimental occurrence is tentatively ascribed to a not uniform contact resistance distribution along the channel width. The subsequent implications on the carrier mobility evaluation are discussed too