Simon T. Ball

Identification of an imprinting control region affecting the expression of all transcripts in the Gnas cluster

Genomic imprinting results in allele-specific silencing according to parental origin. Silencing is brought about by imprinting control regions (ICRs) that are differentially marked in gametogenesis. The group of imprinted transcripts in the mouse Gnas cluster (Nesp, Nespas, Gnasxl, Exon 1A and Gnas) provides a model for analyzing the mechanisms of imprint regulation. We previously identified an ICR that specifically regulates the tissue-specific imprinted expression of the Gnas gene. Here we identify a second ICR at the Gnas cluster. We show that a paternally derived targeted deletion of the germline differentially methylated region (DMR) associated with the antisense Nespas transcript unexpectedly affects both the expression of all transcripts in the cluster and methylation of two DMRs. Our results establish that the Nespas DMR is the principal ICR at the Gnas cluster and functions bidirectionally as a switch for modulating expression of the antagonistically acting genes Gnasxl and Gnas. Uniquely, the Nespas DMR acts on the downstream ICR at exon 1A to regulate tissue-specific imprinting of the Gnas gene.

A cis-acting control region is required exclusively for the tissue-specific imprinting of Gnas

Genomic imprinting brings about allele-specific silencing according to parental origin. Silencing is controlled by cis-acting regulatory regions that are differentially marked during gametogenesis and can act over hundreds of kilobases to silence many genes. Two candidate imprinting control regions (ICRs) have been identified at the compact imprinted Gnas cluster on distal mouse chromosome 2, one at exon 1A upstream of Gnas itself and one covering the promoters for Gnasxl and the antisense Nespas (ref. 8). This imprinted cluster is complex, containing biallelic, maternally and paternally expressed transcripts that share exons. Gnas itself is mainly biallelically expressed but is weakly paternally repressed in specific tissues. Here we show that a paternally derived targeted deletion of the germline differentially methylated region at exon 1A abolishes tissue-specific imprinting of Gnas. This rescues the abnormal phenotype of mice with a maternally derived Gnas mutation. Imprinting of alternative transcripts, Nesp, Gnasxl and Nespas (ref. 13), in the cluster is unaffected. The results establish that the differentially methylated region at exon 1A contains an imprinting control element that specifically regulates Gnas and comprises a characterized ICR for a gene that is only weakly imprinted in a minority of tissues. There must be a second ICR regulating the alternative transcripts.

Meta-Analysis of Calcineurin-Inhibitor-Sparing Regimens in Kidney Transplantation

Calcineurin-inhibitor-sparing strategies in kidney transplantation may spare patients the adverse effects of these drugs, but the efficacy of these strategies is unknown. Here, we conduct a meta-analysis to assess outcomes associated with reducing calcineurin inhibitor exposure from the time of transplantation. We search Medline, Embase, and Cochrane Register of Controlled Trials for randomized controlled trials published between 1966 and 2010 that compared de novo calcineurin-inhibitor-sparing regimens to calcineurin-inhibitor-based regimens. In this analysis, we include 56 studies comprising data from 11337 renal transplant recipients. Use of the contemporary agents belatacept or tofacitinib, in combination with mycophenolate, decreased the odds of overall graft failure (OR 0.61; 95% CI 0.39-0.96; P = 0.03). Similarly, minimization of calcineurin inhibitors in combination with various induction and adjunctive agents reduces the odds of graft failure (OR 0.73; 95% CI 0.58-0.92; P = 0.009). Conversely, the use of inhibitors of mammalian target of rapamycin (mTOR), in combination with mycophenolate, increases the odds of graft failure (OR 1.43; 95% CI 1.08-1.90; P = 0.01). Calcineurin-inhibitor-sparing strategies are associated with less delayed graft function (OR 0.89; 95% CI 0.80-0.98; P = 0.02), improved graft function, and less new-onset diabetes. The more contemporary protocols did not seem to increase rates of acute rejection. In conclusion, this meta-analysis suggests that reducing exposure to calcineurin inhibitors immediately after kidney transplantation may improve clinical outcomes.

Uncoupling antisense-mediated silencing and DNA methylation in the imprinted Gnas cluster.

There is increasing evidence that non-coding macroRNAs are major elements for silencing imprinted genes, but their mechanism of action is poorly understood. Within the imprinted Gnas cluster on mouse chromosome 2, Nespas is a paternally expressed macroRNA that arises from an imprinting control region and runs antisense to Nesp, a paternally repressed protein coding transcript. Here we report a knock-in mouse allele that behaves as a Nespas hypomorph. The hypomorph mediates down-regulation of Nesp in cis through chromatin modification at the Nesp promoter but in the absence of somatic DNA methylation. Notably there is reduced demethylation of H3K4me3, sufficient for down-regulation of Nesp, but insufficient for DNA methylation; in addition, there is depletion of the H3K36me3 mark permissive for DNA methylation. We propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby Nespas modulates demethylation of H3K4me3 resulting in repression of Nesp followed by DNA methylation. This study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of DNA methylation. These results have broad implications for understanding the hierarchy of events in epigenetic silencing by macroRNAs.

DEATH OF MOUSE EMBRYOS THAT LACK A FUNCTIONAL GENE FOR GLUCOSE PHOSPHATE ISOMERASE

A null allele of the Gpi-1s structural gene, that encodes glucose phosphate isomerase (GPI-1; E.C. 5.3.1.9), arose in a mutation experiment and was designated Gpi-1sa-m1H. The viability of homozygotes has been investigated. No offspring homozygous for the null allele were produced by intercrossing two heterozygotes, so the homozygous condition was presumed to be embryonic lethal. Embryos were produced by crossing Gpi-1sa/null heterozygous females and Gpi-1sb/null heterozygous males. Homozygous null embryos were identified at different stages of development by electrophoresis and staining either for GPI-1 alone or GPI-1 plus phosphoglycerate kinase (PGK) activity. At 6 1/2 and 7 1/2 days post coitum homozygous null embryos were present at approximately the expected 25% frequency (37/165; 22.4% overall) although at 7 1/2 days the homozygous null embryos tended to be small. By 8 1/2 days most homozygous null embryos were developmentally retarded and had not developed significantly further than at 7 1/2 days; some were dead or dying. By 9 1/2 days the homozygous null conceptus was characterised by a small implantation site that contained trophoblast and often a small amount of extraembryonic membrane. Surviving trophoblast tissue was also detectable at 10 1/2 days. Previous studies have shown that oocyte-coded GPI-1 persists only until 5 1/2 or 6 1/2 days. Survival of homozygous null embryos to 7 1/2 or 8 1/2 days and survival of certain extraembryonic tissue to 10 1/2 days suggests that the homozygous null condition may not be cell-lethal although it is certainly embryo-lethal. Mutant cells that are deficient in glycolysis may use the pentose phosphate shunt to bypass the block in glycolysis created by the deficiency of glucose phosphate isomerase, and/or might be rescued by the transport, from the maternal blood, of energy sources other than glucose (such as glutamine). Either strategy may only permit slow cell growth that would not be adequate to support normal embryogenesis. Transport of maternal nutrients would be more efficient to the trophoblast and extraembryonic membranes and this may help to explain why these tissues survive for longer than the embryo itself. The morphological similarity between homozygous nulls and androgenetic conceptuses, where the trophoblast also survives better than the embryo, is discussed.

Loss of Gnas imprinting differentially affects REM/NREM sleep and cognition in mice.

It has been suggested that imprinted genes are important in the regulation of sleep. However, the fundamental question of whether genomic imprinting has a role in sleep has remained elusive up to now. In this work we show that REM and NREM sleep states are differentially modulated by the maternally expressed imprinted gene Gnas. In particular, in mice with loss of imprinting of Gnas, NREM and complex cognitive processes are enhanced while REM and REM–linked behaviors are inhibited. This is the first demonstration that a specific overexpression of an imprinted gene affects sleep states and related complex behavioral traits. Furthermore, in parallel to the Gnas overexpression, we have observed an overexpression of Ucp1 in interscapular brown adipose tissue (BAT) and a significant increase in thermoregulation that may account for the REM/NREM sleep phenotypes. We conclude that there must be significant evolutionary advantages in the monoallelic expression of Gnas for REM sleep and for the consolidation of REM–dependent memories. Conversely, biallelic expression of Gnas reinforces slow wave activity in NREM sleep, and this results in a reduction of uncertainty in temporal decision-making processes.

Assessing and comparing rival definitions of delayed renal allograft function for predicting subsequent graft failure.

Background. The traditional definition of delayed graft function (DGF) rests on dialysis requirement during the first postoperative week. Subsequently, a more objective and “functional” definition of DGF (fDGF) has been proposed as an alternative to this dialysis-based definition of DGF (dDGF) and defined as a failure of the serum creatinine to decrease by at least 10% daily on 3 successive days during the first week posttransplantation, irrespective of dialysis requirement. However, an association between fDGF and long-term graft failure has not been fully established, and it is unknown whether fDGF is a better marker of subsequent outcomes than dDGF. Methods. We studied 750 adult deceased donor kidney transplant recipients (1996–2006) and analyzed the association between these two DGF definitions and long-term graft outcome. Results. Univariable associations with death-censored graft failure were seen for both dDGF and fDGF (hazard ratio [HR] 1.59; 95% confidence interval [CI] 1.16–2.18; P=0.004 and HR 1.72; 95% CI 1.26–2.36; P=0.001, respectively). On bivariable analysis (dDGF vs. fDGF), dDGF lost significance, whereas the effect of fDGF persisted (HR 1.52; 95%CI 1.03–2.25; P=0.04). This was also the case in a multivariable model, where fDGF but not dDGF was significantly associated with graft failure (HR 1.47; 95%CI 1.06–2.03; P=0.02). Results were similar for overall graft failure. Conclusions. This study confirms the utility of fDGF as an early marker of subsequent inferior allograft outcomes, suggesting superiority over the traditional (often subjective) dialysis-based definition. Wider adoption of the fDGF definition should be considered, both as a risk-stratification tool in clinical practice and a clinical trial endpoint.

A high-resolution linkage map of the tight skin 2 (Tsk2) locus: A mouse model for scleroderma (SSc) and other cutaneous fibrotic diseases

Tsk2/+ is a novel mutation that first appeared in the offspring of a male from the 101/H strain that had been administered the mutagenic agent, ethylnitrosourea. The mouse was recognized because of the tight skin in the interscapular region (Peters and Ball 1986). In contrast to the Tsk mutation, which is known to reside on Chromosome (Chr) 2 (Green et al. 1976; Siracusa et al. 1993), the Tsk2 mutation has been localized to mouse Chr 1 by linkage studies to two visible Chr 1 mutations (Peters and Ball 1986) and three

A quantitative survey of Western Muslim attitudes to solid organ donation.

Background. It is imperative for healthcare providers to examine Western Muslim attitudes on organ donation, because they are reluctant donors. We explored such opinion with the aid of a quantitative survey. Methods. Voluntary completion of an anonymous survey was promoted (online and paper sampling). For a population target of approximately 1.6 billion, we targeted a completed sample size of 664 to achieve 5% error margins and 99% confidence intervals (assuming 50% response distribution). Logistic regression analysis was performed to assess independent predictors for organ donation approval. Results. In total, 891 global Muslims took the survey with 728 full completions (81.7% completion rate). Paper survey (14% of total) response rate was 62% (124 completed/200 distributed). Western Muslims comprised 76% of participants (n=675) and formed the basis of the analysis. A total of 68.5% of Western Muslims agreed with organ donation, but just 39.3% believed it was compatible with Islam (only 12.7% were registered donors). A total of 1.9% would refuse an organ transplant if required, with 72.4% happy to receive and 25.7% undecided. The main constraints cited by Western Muslims were interpretation of religious scripture (76.5%) and advice from local mosque (70.2%). Predictors for organ donation approval among all global Muslims included younger age, lesser degree of self-rated religiosity, awareness of organ shortages, higher education, and knowing someone with kidney disease/dialysis (all P<0.05). Conclusion. Concern exists among Western Muslims regarding organ donation. Our speculative work should form the basis of larger and more representative assessment of global Muslims to facilitate targeted initiatives to raise awareness.

Mapping studies of the distal imprinting region of mouse chromosome 2.

The known limits of the distal imprinting region of mouse Chromosome (Chr) 2 are defined by the breakpoints of the translocations T(2;8)2Wa, (T2Wa), and T(2;16)28H, (T28H), in distal H3, and proximal H4 respectively. We have shown that T2Wa and T(2;4)1Go, (T1Go), which has a breakpoint in central H3 map close to a, non-agouti. Ada, adenosine deaminase, lies very near the proximal boundary and Ra, ragged, maps very close to the distal boundary, and is less than 0.2 cM from wasted, wst. From the current data Ada can be taken as the proximal, and Ra as the distal gene marker of the imprinting region on the linkage map. From consensus maps twenty three other markers, including fourteen genes, lie between Ada and Ra, some of which may be useful in investigations of imprinting. Of the markers included in the study reported here, four, Ada, ls, lethal spotting, Ra and wst lie or probably lie within the region but none display any evidence of imprinting. We suggest that recombination frequency is elevated in distal Chr 2, because in none of the crosses could the most closely linked marker be ordered in relation to the translocation breakpoint due to the high frequency of double crossovers.