Publications

Functionally significant heterozygous mutations in the Melanocortin-4 receptor (MC4R) have been implicated in 2.5% of early onset obesity cases in European cohorts. The role of mutations in this gene in severely obese adults, particularly in smaller North American patient cohorts, has been less convincing. More recently, it has been proposed that mutations in a phylogenetically and physiologically related receptor, the Melanocortin-3 receptor (MC3R), could also be a cause of severe human obesity. The objectives of this study were to determine if mutations impairing the function of MC4R or MC3R were associated with severe obesity in North American adults. We studied MC4R and MC3R mutations detected in a total of 1821 adults (889 severely obese and 932 lean controls) from two cohorts. We systematically and comparatively evaluated the functional consequences of all mutations found in both MC4R and MC3R. The total prevalence of rare MC4R variants in severely obese North American adults was 2.25% (CI(95%): 1.44-3.47) compared with 0.64% (CI(95%): 0.26-1.43) in lean controls (P < 0.005). After classification of functional consequence, the prevalence of MC4R mutations with functional alterations was significantly greater when compared with controls (P < 0.005). In contrast, the prevalence of rare MC3R variants was not significantly increased in severely obese adults [0.67% (CI(95%): 0.27-1.50) versus 0.32% (CI(95%): 0.06-0.99)] (P = 0.332). Our results confirm that mutations in MC4R are a significant cause of severe obesity, extending this finding to North American adults. However, our data suggest that MC3R mutations are not associated with severe obesity in this population.

We applied genome-wide profiling to successive salt-extracted fractions of micrococcal nuclease-treated Drosophila chromatin. Chromatin fractions extracted with 80 mM or 150 mM NaCl after digestion contain predominantly mononucleosomes and represent classical "active" chromatin. Profiles of these low-salt soluble fractions display phased nucleosomes over transcriptionally active genes that are locally depleted of histone H3.3 and correspond closely to profiles of histone H2Av (H2A.Z) and RNA polymerase II. This correspondence suggests that transcription can result in loss of H3.3+H2Av nucleosomes and generate low-salt soluble nucleosomes. Nearly quantitative recovery of chromatin is obtained with 600 mM NaCl; however, the remaining insoluble chromatin is enriched in actively transcribed regions. Salt-insoluble chromatin likely represents oligonucleosomes that are attached to large protein complexes. Both low-salt extracted and insoluble chromatin are rich in sequences that correspond to epigenetic regulatory elements genome-wide. The presence of active chromatin at both extremes of salt solubility suggests that these salt fractions capture bound and unbound intermediates in active processes, thus providing a simple, powerful strategy for mapping epigenome dynamics.

Type I interferons, a protein family of multiple IFNalphas and a single IFNbeta, initially identified on the basis of their antiviral activities have recently been attributed important roles in bacterial and parasitic infections. To assess the cellular sources of IFNbeta, the IFN produced first in most situations, we created an IFNbeta reporter-knockin mouse, in which yellow fluorescent protein (YFP) is expressed from a bicistronic mRNA linked by an internal ribosomal entry site to the endogenous IFNbeta mRNA. This YFP expression allows spatiotemporal tracking of the initiation of the type I IFN response on a single-cell level. In vitro bone marrow-derived macrophages (BMMPhis) and bone marrow-derived dendritic cells (BMDCs) show IFNbeta production from distinct cell subpopulations in response to defined pathogen compounds. A subpopulation of GMCSF-derived BMDCs produced IFNbeta after poly(I:C), 3'5'-cytidylylguanosine (CpG), or LPS treatment, whereas Flt3-L-cultured plasmacytoid DCs (pDCs) responded mainly to CpG. After poly(I:C) injection in vivo, IFNbeta-producing cells localize to the splenic marginal zone and the lymph node subcapsular sinus. Infection with murine cytomegalovirus (MCMV) induces IFNbeta/YFP expression exclusively in few activated pDCs at the T cell/B cell interface of the splenic white pulp. This IFNbeta/YFP reporter mouse represents a reliable tool for the visualization and characterization of IFNbeta-producing cells in vitro and in vivo.

PURPOSE

This open-label, prospective, single-arm, phase II study combined erlotinib with radiation therapy (XRT) and temozolomide to treat glioblastoma multiforme (GBM) and gliosarcoma. The objectives were to determine efficacy of this treatment as measured by survival and to explore the relationship between molecular markers and treatment response.

PATIENTS AND METHODS

Sixty-five eligible adults with newly diagnosed GBM or gliosarcoma were enrolled. We intended to treat patients not currently treated with enzyme-inducing antiepileptic drugs (EIAEDs) with 100 mg/d of erlotinib during XRT and 150 mg/d after XRT. Patients receiving EIAEDs were to receive 200 mg/d of erlotinib during XRT and 300 mg/d after XRT. After XRT, the erlotinib dose was escalated until patients developed tolerable grade 2 rash or until the maximum allowed dose was reached. All patients received temozolomide during and after XRT. Molecular markers of epidermal growth factor receptor (EGFR), EGFRvIII, phosphatase and tensin homolog (PTEN), and methylation status of the promotor region of the MGMT gene were analyzed from tumor tissue. Survival was compared with outcomes from two historical phase II trials.

RESULTS

Median survival was 19.3 months in the current study and 14.1 months in the combined historical control studies, with a hazard ratio for survival (treated/control) of 0.64 (95% CI, 0.45 to 0.91). Treatment was well tolerated. There was a strong positive correlation between MGMT promotor methylation and survival, as well as an association between MGMT promotor-methylated tumors and PTEN positivity shown by immunohistochemistry with improved survival.

CONCLUSION

Patients treated with the combination of erlotinib and temozolomide during and following radiotherapy had better survival than historical controls. Additional studies are warranted.