A systematic review of high impact CpG sites and regions for MGMT methylation in glioblastoma [A systematic review of MGMT methylation in GBM].

BACKGROUND: MGMT (O 6 -methylguanine-DNA methyltransferase) promoter methylation is a commonly assessed prognostic marker in glioblastoma (GBM). Epigenetic silencing of the MGMT gene by promoter methylation is associated with greater overall and progression free survival with alkylating agent regimens. To date, there is marked heterogeneity in how MGMT promoter methylation is tested and which CpG sites are interrogated. METHODS: To further elucidate which MGMT promoter CpG sites are of greatest interest, we performed comprehensive searches in PubMed, Web of Science, and Embase and reviewed 2,925 article abstracts. We followed the GRADE scoring system to assess risk of bias and the quality of the studies we included. RESULTS: We included articles on adult glioblastoma that examined significant sites or regions within MGMT promoter for the outcomes: overall survival, progression free survival, and/or MGMT expression. We excluded systemic reviews and articles on lower grade glioma. fifteen articles met inclusion criteria with variable overlap in laboratory and statistical methods employed, as well as CpG sites interrogated. Pyrosequencing or BeadChip arrays were the most popular methods utilized, and CpG sites between CpG's 70-90 were most frequently investigated. Overall, there was moderate concordance between the CpG sites that the studies reported to be highly predictive of prognosis. Combinations or means of sites between CpG's 73-89 were associated with improved OS and PFS. Six studies identified CpG sites associated with prognosis that were closer to the transcription start site: CpG's 8, 19, 22, 25, 27, 32,38, and CpG sites 21-37, as well as low methylation level of the enhancer regions. CONCLUSION: The following systematic review details a comprehensive investigation of the current literature and highlights several potential key CpG sites that demonstrate significant association with OS, PFS, and MGMT expression. However, the relationship between extent of MGMT promoter methylation and survival may be non-linear and could be influenced by potential CpG hotspots, the extent of methylation at each CpG site, and MGMT enhancer methylation status. There were several limitations within the studies such as smaller sample sizes, variance between methylation testing methods, and differences in the various statistical methods to test for association to outcome. Further studies of high impact CpG sites in MGMT methylation is warranted.

LNK/SH2B3 as a novel driver in juvenile myelomonocytic leukemia.

Mutations in five canonical Ras pathway genes (NF1, NRAS, KRAS, PTPN11 and CBL) are detected in nearly 90% of patients with juvenile myelomonocytic leukemia (JMML), a frequently fatal malignant neoplasm of early childhood. In this report, we describe seven patients diagnosed with SH2B3-mutated JMML, including five patients who were found to have initiating, loss of function mutations in the gene. SH2B3 encodes the adaptor protein LNK, a negative regulator of normal hematopoiesis upstream of the Ras pathway. These mutations were identified to be germline, somatic or a combination of both. Loss of function of LNK, which has been observed in other myeloid malignancies, results in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of the JAK/STAT signaling pathway. In vitro studies of induced pluripotent stem cell-derived JMML-like hematopoietic progenitor cells (HPCs) also demonstrated sensitivity of SH2B3- mutated HPCs to JAK inhibition. Lastly, we describe two patients with JMML and SH2B3 mutations who were treated with the JAK1/2 inhibitor ruxolitinib. This report expands the spectrum of initiating mutations in JMML and raises the possibility of targeting the JAK/STAT pathway in patients with SH2B3 mutations.

Molecular assessment of pretransplant chemotherapy in the treatment of juvenile myelomonocytic leukemia.

BACKGROUND: Despite the intensity of hematopoietic stem cell transplantation (HCT), relapse remains the most common cause of death in juvenile myelomonocytic leukemia (JMML). In contrast to other leukemias where therapy is used to reduce leukemic burden prior to transplant, many patients with JMML proceed directly to HCT with active disease. The objective of this study was to elucidate whether pre-HCT therapy has an effect on the molecular burden of disease and how this affects outcome post-HCT. PROCEDURE: Twenty-one patients with JMML who received pre-HCT therapy and were transplanted at UCSF were analyzed in this study. The mutant allele frequency of the driver mutation was assessed before and after pre-HCT therapy, using custom amplicon next-generation sequencing. RESULTS: Of the 21 patients, seven patients (33%) responded to therapy with a significant reduction in their mutant allele frequency and were classified as molecular responders. Six of these patients received moderate-intensity chemotherapy, one patient received only azacitidine. The 5-year progression-free survival after HCT of molecular responders was 100% versus 61% for nonresponders (P = .12). Survival of molecular nonresponders was not improved by use of high-intensity conditioning, but patients were salvaged if they experienced severe graft versus host disease. There were no baseline clinical characteristics that were associated with response to pre-HCT therapy. CONCLUSIONS: Despite the myelodysplastic nature of JMML, patients treated with pre-HCT therapy can achieve molecular remissions. These patients experienced a trend toward improved outcomes post-HCT. Importantly, molecular testing can be helpful to distinguish between responders and nonresponders and should become an integral part of clinical care.

Upregulation of cholesterol 24-hydroxylase following hypoxia-ischemia in neonatal mouse brain.

BackgroundMaintenance of cholesterol homeostasis is crucial for brain development. Brain cholesterol relies on de novo synthesis and is cleared primarily by conversion to 24S-hydroxycholesterol (24S-HC) with brain-specific cholesterol 24-hydroxylase (CYP46A1). We aimed to investigate the impact of hypoxia-ischemia (HI) on brain cholesterol metabolism in the neonatal mice.MethodsPostnatal day 9 C57BL/6 pups were subjected to HI using the Vannucci model. CYP46A1 expression was assessed with western blotting and its cellular localization was determined using immunofluorescence staining. The amount of brain cholesterol, 24S-HC in the cortex and in the serum, was measured with enzyme-linked immunosorbent assay (ELISA).ResultsThere was a transient cholesterol loss at 6 h after HI. CYP46A1 was significantly upregulated at 6 and 24 h following HI with a concomitant increase of 24S-HC in the ipsilateral cortex and in the serum. The serum levels of 24S-HC correlated with those in the brain, as well as with necrotic and apoptotic cell death evaluated by the expression of spectrin breakdown products and cleaved caspase-3 at 6 and 24 h after HI.ConclusionEnhanced cholesterol turnover by activation of CYP46A1 represents disrupted brain cholesterol homeostasis early after neonatal HI. 24S-HC might be a novel blood biomarker for severity of hypoxic-ischemic encephalopathy with potential clinical application.

FoxO4 interacts with the sterol regulatory factor SREBP2 and the hypoxia inducible factor HIF2α at the CYP51 promoter.

The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule. A key enzymatic reaction, which occurs at the top of the Bloch and Kandutsch-Russell pathways, is the demethylation of lanosterol and dihydrolanosterol (DHL). This reaction is catalyzed by lanosterol 14α demethylase (CYP51) and requires three oxygen molecules. Thus, it is the first step in the distal pathway to be susceptible to oxygen deprivation. Having previously identified that the forkhead transcription factor 4 (FoxO4) represses CYP51 expression, we aimed to characterize its role at the CYP51 promoter. Hypoxia-treated 3T3L1 cells showed decreased cholesterol biosynthesis, accumulation of lanosterol/DHL, and stimulation of FoxO4 expression and its cytoplasmic translocation to the nucleus. Transfection assays with a CYP51 promoter reporter gene revealed that FoxO4 and sterol regulatory element binding protein (SREBP)2 exert a stimulatory effect, whereas FoxO4 and the hypoxia inducible factor (HIF)2α repress CYP51 promoter activity. Electromobility shift, chromatin immunoprecipitation, pull-down, and coimmunoprecipitation assays show that FoxO4 interacts with SREBP2 and HIF2α to modulate CYP51 promoter activity. We also show an inverse correlation between FoxO4 and CYP51 in adipose tissue of ob/ob mice and mouse fetal cortical neurons exposed to hypoxia. Overall, these studies demonstrate a role for FoxO4 in the regulation of CYP51 expression.

Effects of FoxO4 overexpression on cholesterol biosynthesis, triacylglycerol accumulation, and glucose uptake.

The Forkhead transcription factors FoxO1, FoxO3a, and FoxO4 play a prominent role in regulating cell survival and cell cycle. Whereas FOXO1 was shown to mediate insulin sensitivity and adipocyte differentiation, the role of the transcription factor FoxO4 in metabolism remains ill defined. To uncover the effects of FoxO4, we generated a cellular model of stable FoxO4 overexpression and subjected it to microarray-based gene expression profiling. While pathway analysis revealed a disruption of cholesterol biosynthesis gene expression, biochemical studies revealed an inhibition of cholesterol biosynthesis, which was coupled with decreased mRNA levels of lanosterol 14alpha demethylase (CYP51). FoxO4-mediated repression of CYP51 led to the accumulation of 24,25 dihydrolano-sterol (DHL), which independently and unlike lanosterol inhibited cholesterol biosynthesis. Furthermore, FoxO4-overexpressing cells accumulated lipid droplets and triacylglycerols and had an increase in basal glucose uptake. Recapitulation of these effects was obtained following treatment with CYP51 inhibitors, which also induce DHL buildup. Moreover, DHL but not lanosterol strongly stimulated liver X receptor alpha (LXRalpha) activity, suggesting that DHL and LXRalpha mediate the downstream effects initiated by FoxO4. Together, these studies suggest that FoxO4 acts on CYP51 to regulate the late steps of cholesterol biosynthesis.

Obesity and lipodystrophy--where do the circles intersect?

Adipose tissue is unique in that it can undergo significant hypertrophy and atrophy, resulting in wide ranges of obesities and lipodystrophies. At the base of this elasticity is the lipid-filled adipocyte, which can either overfill by storing large amounts of triglycerides or shrink to a tiny cell by depleting its lipids and as such is remarkable in sustaining insults. As a major energy reservoir, the adipocyte may hold considerable calories necessary for survival and reproduction, two functions that are essential for the survival of the species. This review will summarize some of the recent studies that have advanced our understanding of the central and peripheral mechanisms that are initiated by adipocyte-secreted factors such as leptin, adiponectin, resistin, and retinol-binding protein 4. The intersection of obesity and lipodystrophy results in insulin resistance, which may be unlocked by elucidating the roles of these factors in pathways that control insulin sensitivity and glucose uptake.

Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution.

Leptin and reproduction.

Leptin, a hormone secreted from adipose tissue, plays an important role in reproductive physiology. It has been shown to stimulate the reproductive system by rescuing the sterility of leptin-deficient mice and advancing the onset of puberty in normal mice. Although leptin is not critical for the biology of pregnancy in mice, its ability to reduce food intake is blunted in mid-gestation suggesting that late pregnancy may be a leptin-resistant state. Modifier genes originating from the Balb/cJ genetic background profoundly alter the sterile-obese phenotype of ob/ob mice by reducing their obesity and stimulating their reproductive system despite the absence of leptin. The mechanism of leptin's action on the reproductive system remains to be determined but is likely to be mediated by multiple factors.

20 years of leptin: leptin and reproduction: past milestones, present undertakings, and future endeavors.

The association between leptin and reproduction originated with the leptin-mediated correction of sterility in ob/ob mice and initiation of reproductive function in normal female mice. The uncovering of a central leptin pathway regulating food intake prompted the dissection of neuroendocrine mechanisms involving leptin in the metabolic control of reproduction. The absence of leptin receptors on GnRH neurons incited a search for intermediary neurons situated between leptin-responsive and GnRH neurons. This review addresses the most significant findings that have furthered our understanding of recent progress in this new field. The role of leptin in puberty was impacted by the discovery of neurons that co-express kisspeptin, neurokinin B, and dynorphin and these could act as leptin intermediates. Furthermore, the identification of first-order leptin-responsive neurons in the premammilary ventral nucleus and other brain regions opens new avenues to explore their relationship to GnRH neurons. Central to these advances is the unveiling that agouti-related protein/neuropeptide Y neurons project onto GnRH and kisspeptin neurons, allowing for a crosstalk between food intake and reproduction. Finally, while puberty is a state of leptin sensitivity, mid-gestation represents a state of leptin resistance aimed at building energy stores to sustain pregnancy and lactation. The mechanisms underlying leptin resistance in pregnancy have lagged; however, the establishment of this natural state is significant. Reproduction and energy balance are tightly controlled and backed up by redundant mechanisms that are critical for the survival of our species. It will be the goal of the following decade to shed new light on these complex and essential pathways.

First report of a deletion encompassing an entire exon in the homogentisate 1,2-dioxygenase gene causing alkaptonuria.

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two ß-pleated sheets encoded by exon 2 were missing in the mutant structure, other ß-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

Duplication of C7orf58, WNT16 and FAM3C in an obese female with a t(7;22)(q32.1;q11.2) chromosomal translocation and clinical features resembling Coffin-Siris Syndrome.

We characterized the t(7;22)(q32;q11.2) chromosomal translocation in an obese female with coarse features, short stature, developmental delay and a hypoplastic fifth digit. While these clinical features suggest Coffin-Siris Syndrome (CSS), we excluded a CSS diagnosis by exome sequencing based on the absence of deleterious mutations in six chromatin-remodeling genes recently shown to cause CSS. Thus, molecular characterization of her translocation could delineate genes that underlie other syndromes resembling CSS. Comparative genomic hybridization microarrays revealed on chromosome 7 the duplication of a 434,682 bp region that included the tail end of an uncharacterized gene termed C7orf58 (also called CPED1) and spanned the entire WNT16 and FAM3C genes. Because the translocation breakpoint on chromosome 22 did not disrupt any apparent gene, her disorder was deemed to result from the rearrangement on chromosome 7. Mapping of yeast and bacterial artificial chromosome clones by fluorescent in situ hybridization on chromosome spreads from this patient showed that the duplicated region and all three genes within it were located on both derivative chromosomes 7 and 22. Furthermore, DNA sequencing of exons and splice junctional regions from C7orf58, WNT16 and FAM3C revealed the presence of potential splice site and promoter mutations, thereby augmenting the detrimental effect of the duplicated genes. Hence, dysregulation and/or disruptions of C7orf58, WNT16 and FAM3C underlie the phenotype of this patient, serve as candidate genes for other individuals with similar clinical features and could provide insights into the physiological role of the novel gene C7orf58.

Homozygous deletion of six olfactory receptor genes in a subset of individuals with Beta-thalassemia.

Progress in the functional studies of human olfactory receptors has been largely hampered by the lack of a reliable experimental model system. Although transgenic approaches in mice could characterize the function of individual olfactory receptors, the presence of over 300 functional genes in the human genome becomes a daunting task. Thus, the characterization of individuals with a genetic susceptibility to altered olfaction coupled with the absence of particular olfactory receptor genes will allow phenotype/genotype correlations and vindicate the function of specific olfactory receptors with their cognate ligands. We characterized a 118 kb ß-globin deletion and found that its 3' end breakpoint extends to the neighboring olfactory receptor region downstream of the ß-globin gene cluster. This deletion encompasses six contiguous olfactory receptor genes (OR51V1, OR52Z1, OR51A1P, OR52A1, OR52A5, and OR52A4) all of which are expressed in the brain. Topology analysis of the encoded proteins from these olfactory receptor genes revealed that OR52Z1, OR52A1, OR52A5, and OR52A4 are predicted to be functional receptors as they display integral characteristics of G-proteins coupled receptors. Individuals homozygous for the 118 kb ß-globin deletion are afflicted with ß-thalassemia due to a homozygous deletion of the ß-globin gene and have no alleles for the above mentioned olfactory receptors genes. This is the first example of a homozygous deletion of olfactory receptor genes in human. Although altered olfaction remains to be ascertained in these individuals, such a study can be carried out in ß-thalassemia patients from Malaysia, Indonesia and the Philippines where this mutation is common. Furthermore, OR52A1 contains a γ-globin enhancer, which was previously shown to confer continuous expression of the fetal γ-globin genes. Thus, the hypothesis that ß-thalassemia individuals, who are homozygous for the 118 kb deletion, may also have an exacerbation of their anemia due to the deletion of two copies of the γ-globin enhancer element is worthy of consideration.

Overexpression of the transcription factor Foxo4 is associated with rapid glucose clearance.

Leptin treatment ameliorates lipoatrophic diabetes in animal models and humans. Transgenic mice overexpressing leptin (LepTg) are lipoatrophic but not diabetic and thus represent a model for elucidating mechanisms of leptin-mediated glucose homeostasis. In this communication, we show that LepTg mice overexpress the forkhead transcription factor foxo4 in their remnant adipose tissue. To further characterize the role of foxo4 in adipose tissue, we generated transgenic mice overexpressing a constitutive active form of foxo4 (A3foxo4) under the control of the aP2 promoter/enhancer. aP2-A3foxo4 mice are not lipoatrophic but are able to clear glucose rapidly similar to LepTg mice. In addition, both LepTg and A3foxo4 mice show in their adipocytes increased AMP-activated protein kinase (AMPK) phosphorylation, suggesting a link between AMPK, glucose clearance, foxo4 and the leptin axis. These studies shed new light on mechanisms by which leptin treatment improves glucose disposal.

Deletion of the serotonin 2c receptor from transgenic mice overexpressing leptin does not affect their lipodystrophy but exacerbates their diet-induced obesity.

The binding of leptin to hypothalamic neurons elicits inhibition of orexigenic NPY/AgRP neurons and stimulation of anorexigenic POMC/CART neurons. Projections of serotonergic neurons onto POMC neurons suggest that leptin and serotonin converge onto POMC neurons to regulate body weight. We probed the interaction of these pathways by generating transgenic mice overexpressing leptin (LepTg) without 5HT2c receptors. On a chow diet, the lean phenotype of LepTg mice was unaffected by the absence of 5HT2c receptors, whereas on a high fat diet, LepTg/5HT2c receptors knockout mice showed an exacerbation of diet-induced obesity. POMC mRNA levels were low in LepTg, 5HT2c receptors knockout and LepTg/5HT2c receptors knockout mice, demonstrating that perturbations of the 5HT2c receptor and leptin pathways, either alone or in combination, negatively impact on POMC expression. Thus, on a chow diet, leptin action is independent of 5HT2c receptors whereas on a high fat diet 5HT2c receptors are required for the attenuation of obesity.

Mosaicism for a full mutation, premutation, and deletion of the CGG repeats results in 22% FMRP and elevated FMR1 mRNA levels in a high-functioning fragile X male.

The molecular basis in the majority of fragile X patients results from expansion of the CGG repeats in the FMR1 gene causing its transcriptional silencing and deficiency of its encoded protein FMRP. In this communication, we report on a male patient who lacks the characteristic physical features of fragile X and carries a fully methylated mutation, a premutation, a non-methylated full mutation, and a microdeletion encompassing the entire CGG repeat region and 42 bp of upstream flanking sequence. Southern blot analysis revealed that the methylated full mutation accounted for only 10% of his genotype while the premutation/non-methylated full mutation and the microdeletion constituted 37% and 53%, respectively. Immunofluorescent staining of FMRP demonstrated the presence of 22% FMRP in his peripheral blood leukocytes and quantitative RT-PCR revealed a 3.6-fold elevation of FMR1 mRNA levels. Developmental assessments indicated that while he has a learning disability, he does not have mental retardation. Because previous reports had noted that 28% FMRP expression is associated with a characteristic fragile X phenotype, we propose that in our patient the association of 22% FMRP levels with normal physical features and a high-functioning status may have resulted from increased FMRP stability by a mechanism that takes into account the CGG microdeletion and elevated mRNA levels.

The use of animal models to dissect the biology of leptin.

Our understanding of the effects of leptin have stemmed mainly from animal studies, which continue to leave important clues of its roles in physiology, metabolism, neuroscience, and cell signaling. Since its discovery, leptin has been linked to various pathways, either directly at its primary site of action in the hypothalamus, or indirectly via downstream effector pathways such as in adipocytes and muscle. Leptin's importance is exemplified by the lack of redundant backup mechanisms, since leptin-deficient mice are obese, diabetic, and sterile. Investigations uncovering the pleiotropic actions of leptin were unfolded mainly from rodent models. Thus, this chapter focuses on these studies and, more specifically, on those findings recently brought forward by transgenic mice overexpressing leptin. The vast amount of biology that has been ascribed to leptin encompasses effects on food intake, insulin sensitivity, adiposity, thermogenesis, reproduction, immunity, and bone regulation. Mechanisms underlying leptin's action revolve essentially around neural pathways but also encompass to a lesser extent peripheral mechanisms. The roles of leptin along these axes are reviewed, with particular emphasis on pathways and phenotypes generated by transgenic hyperleptinemia. An evolutionary significance of hyperleptinemia in association with development of leptin resistance is suggested as a protective armament against some of the detrimental effects caused by hyperleptinemia in transgenic mice overexpressing leptin.

Overexpression of leptin in transgenic mice leads to decreased basal lipolysis, PKA activity, and perilipin levels.

Transgenic mice overexpressing leptin (LepTg) exhibit substantial reductions in adipose mass. Since the binding of leptin to its receptor activates the sympathetic nervous system, we reasoned that the lean state of the LepTg mice could be caused by chronic lipolysis. Instead, the LepTg mice exhibited a low basal lipolysis state and their lean phenotype was not dependent on the presence of beta3-adrenergic receptors. In their white adipose tissue, protein levels of protein kinase A, hormone-sensitive lipase, and ADRP were not impaired. However, compared to normal mice, perilipin, perilipin mRNA, and cAMP-stimulated PKA activity were significantly attenuated. Overall, we demonstrate that the lean phenotype of the LepTg mice does not result in a chronically elevated lipolytic state, but instead in a low basal lipolysis state characterized by a decrease in perilipin and PKA activity in white fat.