Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM

O⁶-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient''s subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089–13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301–7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.     

Evolution at the tip and base of the X chromosome in an African population of Drosophila melanogaster

Hitchhiking effects of advantageous mutations have been invoked to explain reduced polymorphism in regions of low crossing-over in Drosophila. Besides reducing DNA heterozygosity, hitchhiking effects should produce strong linkage disequilibrium and a frequency spectrum skewed toward an excess of rare polymorphisms (compared to the neutral expectation). We measured DNA polymorphism in a Zimbabwe population of D. melanogaster at three loci, yellow, achaete, and suppressor of forked, located in regions of reduced crossing-over. Similar to previously published surveys of these genomic regions in other populations, we observed low levels of nucleotide variability. However, the frequency spectrum was compatible with a neutral model, and there was abundant evidence for recombination in the history of the yellow and ac genes. Thus, some aspects of the data cannot be accounted for by a simple hitchhiking model. An alternative hypothesis, background selection, might be compatible with the observed patterns of linkage disequilibrium and the frequency spectrum. However, this model cannot account for the observed reduction in nucleotide heterozygosity. Thus, there is currently no satisfactory theoretical model for the data from the tip and base of the X chromosome in D. melanogaster.      

Infertility in CFW/R1 mice associated with cystic dilatation of the bulbourethral gland

A third of male inbred CFW/R1 mice in a breeding colony developed subcutaneous, bilateral, perineal masses determined to be cystic bulbourethral glands. The masses developed in mice between 4 and 15 months of age. After development of these perineal masses, diseased males were unable to produce offspring. Gross examination revealed the masses impinging on the scrotum and displacing the testes into the inguinal canal. The perineal masses were paired, membranous, translucent cysts, 6 to 10 mm3, attached to the bulbocavernosus muscle and connected to the pelvic urethra by way of a non-patent duct. The cysts contained a clear to tan, minimally cellular, viscous fluid with high mucus content, as documented by examination of Wright Giemsa-stained cytologic preparations. Histologic examination of hematoxylin and eosin-stained sections revealed cystic tubuloalveolar glands surrounded by striated muscle and lined by a single layer of pyramidal cuboidal to columnar epithelial cells with pale, basophilic, lacy cytoplasm and round, basal, condensed nuclei. These gross and histopathologic findings were consistent with cystic dilatation of the bulbourethral gland.     

Requirement of a Src family kinase for initiating calcium release at fertilization in starfish eggs.

Signal transduction leading to calcium release in echinoderm eggs at fertilization requires phospholipase Cgamma-mediated production of inositol trisphosphate (IP(3)), indicating that a tyrosine kinase is a likely upstream regulator. Because previous work has shown a fertilization-dependent association between the Src homology 2 (SH2) domains of phospholipase Cgamma and a Src family kinase, we examined whether a Src family kinase was required for Ca(2+) release at fertilization. To inhibit the function of kinases in this family, we injected starfish eggs with the SH2 domains of Src and Fyn kinases. This inhibited Ca(2+) release in response to fertilization but not in response to injection of IP(3). We further established the specificity of the inhibition by showing that the SH2 domains of several other tyrosine kinases (Abl, Syk, and ZAP-70), and the SH3 domain of Src, were not inhibitory. Also, a point-mutated Src SH2 domain, which has reduced affinity for phosphotyrosine, was a correspondingly less effective inhibitor of fertilization-induced Ca(2+) release. These results indicate that a Src family kinase, by way of its SH2 domain, links sperm-egg interaction to IP(3)-mediated Ca(2+) release at fertilization in starfish eggs.     

Massively Parallel Signature Sequencing and Bioinformatics Analysis Identifies Up-Regulation of TGFBI and SOX4 in Human Glioblastoma

Background

A comprehensive network-based understanding of molecular pathways abnormally altered in glioblastoma multiforme (GBM) is essential for developing effective therapeutic approaches for this deadly disease.

Methodology/Principal Findings

Applying a next generation sequencing technology, massively parallel signature sequencing (MPSS), we identified a total of 4535 genes that are differentially expressed between normal brain and GBM tissue. The expression changes of three up-regulated genes, CHI3L1, CHI3L2, and FOXM1, and two down-regulated genes, neurogranin and L1CAM, were confirmed by quantitative PCR. Pathway analysis revealed that TGF- β pathway related genes were significantly up-regulated in GBM tumor samples. An integrative pathway analysis of the TGF β signaling network identified two alternative TGF−β signaling pathways mediated by SOX4 (sex determining region Y-box 4) and TGFBI (Transforming growth factor beta induced). Quantitative RT-PCR and immunohistochemistry staining demonstrated that SOX4 and TGFBI expression is elevated in GBM tissues compared with normal brain tissues at both the RNA and protein levels. In vitro functional studies confirmed that TGFBI and SOX4 expression is increased by TGF- β stimulation and decreased by a specific inhibitor of TGF- β receptor 1 kinase.

Conclusions/Significance

Our MPSS database for GBM and normal brain tissues provides a useful resource for the scientific community. The identification of non-SMAD mediated TGF−β signaling pathways acting through SOX4 and TGFBI (GENE ID:7045) in GBM indicates that these alternative pathways should be considered, in addition to the canonical SMAD mediated pathway, in the development of new therapeutic strategies targeting TGF−β signaling in GBM. Finally, the construction of an extended TGF- β signaling network with overlaid gene expression changes between GBM and normal brain extends our understanding of the biology of GBM.     

Genetic variation in South Indian castes: evidence from Y-chromosome, mitochondrial, and autosomal polymorphisms

Background

Major population movements, social structure, and caste endogamy have influenced the genetic structure of Indian populations. An understanding of these influences is increasingly important as gene mapping and case-control studies are initiated in South Indian populations.

Results

We report new data on 155 individuals from four Tamil caste populations of South India and perform comparative analyses with caste populations from the neighboring state of Andhra Pradesh. Genetic differentiation among Tamil castes is low (R_ST = 0.96% for 45 autosomal short tandem repeat (STR) markers), reflecting a largely common origin. Nonetheless, caste- and continent-specific patterns are evident. For 32 lineage-defining Y-chromosome SNPs, Tamil castes show higher affinity to Europeans than to eastern Asians, and genetic distance estimates to the Europeans are ordered by caste rank. For 32 lineage-defining mitochondrial SNPs and hypervariable sequence (HVS) 1, Tamil castes have higher affinity to eastern Asians than to Europeans. For 45 autosomal STRs, upper and middle rank castes show higher affinity to Europeans than do lower rank castes from either Tamil Nadu or Andhra Pradesh. Local between-caste variation (Tamil Nadu R_ST = 0.96%, Andhra Pradesh R_ST = 0.77%) exceeds the estimate of variation between these geographically separated groups (R_ST = 0.12%). Low, but statistically significant, correlations between caste rank distance and genetic distance are demonstrated for Tamil castes using Y-chromosome, mtDNA, and autosomal data.

Conclusion

Genetic data from Y-chromosome, mtDNA, and autosomal STRs are in accord with historical accounts of northwest to southeast population movements in India. The influence of ancient and historical population movements and caste social structure can be detected and replicated in South Indian caste populations from two different geographic regions.     

Apparent overdominance of enzyme specific activity in two marine bivalves

Electrophoretic examination of a natural population sample of 332 mussels (Mytilus trossulus) revealed ten active allozyme alleles for the octopine dehydrogenase (Odh) locus and a statistically significant (P<0.005) departure from expected genotypic proportions caused by a deficiency of heterozygous genotypes. In vitro specific activity for octopine dehydrogenase (E.C. 1.5.1.11) was determined for 207 mussels representing 17 different Odh genotypes. Odh heterozygotes had an average specific activity that was 19% greater than that of apparently homozygous genotypes, a significant (P<0.05) difference. Electrophoretic examination of a natural population sample of 209 oysters (Crassostrea virginica) revealed 23 active allozyme alleles for the leucine aminopeptidase-2 (Lap-2) locus and a non-significant (P>0.05) deficiency of heterozygous genotypes. In vitro specific activity for leucine aminopeptidase (E.C. 3.4.-.-) was determined for 89 oysters representing 19 different Lap-2 genotypes. Lap-2 heterozygotes had an average specific activity that was 56% greater than that of homozygous genotypes, a significant (P<0.0001) difference. Possible explanations for the apparent overdominance in enzyme specific activity and the deficiency of heterozygotes include null alleles, molecular imprinting and aneuploidy.     

Sex-specific consequences of life in the city

The vast majority of species are excluded from human-dominatedlandscapes, but some species persist and appear to thrive, despitefacing challenges they would not encounter in less disturbedhabitat. Corticosteroid (cort) secretion may play a significantrole in enabling an animal to cope with urban breeding habitatbecause it helps mediate physiological and behavioral responsesto environmental challenges. Here we present tests of 3 hypothesesrelating to cort secretion in urban animals: 1) environmentalchallenges vary between urban and rural habitat, as reflectedin differing levels of cort secretion in animals breeding inthese habitat types, 2) there are fitness correlates associatedwith variation in cort levels within populations, and 3) parasiteload is one of the mechanistic links between variation in cortlevels and reproductive success. Male white-crowned sparrows(Zonotrichia leucophrys) in urban habitat had significantlyhigher baseline corticosterone levels than males in rural habitat,whereas female Z. leucophrys' hormone levels did not differbetween the 2 habitat types. To assess the fitness correlatesof variation in hormone levels, we monitored seasonal reproductivesuccess in one urban population and found that baseline corticosteronelevels were predictive of female, but not male, reproductivesuccess: females with higher corticosterone fledged fewer offspring.Data do not suggest a direct relationship between corticosteronelevels and fitness and instead indicate an indirect relationshipbetween hormone secretion, territory quality, body condition,and reproductive success. Females with blood parasites had lowerheterophil:lymphocyte ratios, lower body condition scores, fewermates, and fledged fewer offspring than females without parasites.In contrast, parasites did not negatively affect reproductivesuccess in males but were associated with reduced body condition.Corticosterone levels did not differ between birds with andwithout parasites. In tests of all 3 of our hypotheses, we foundsex-specific effects, highlighting the importance of consideringsex in investigation of physiological responses to disturbance.     

HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore

Centromeres of higher eukaryotes are epigenetically marked by the centromere-specific CENP-A nucleosome. New CENP-A recruitment requires the CENP-A histone chaperone HJURP. In this paper, we show that a LacI (Lac repressor) fusion of HJURP drove the stable recruitment of CENP-A to a LacO (Lac operon) array at a noncentromeric locus. Ectopically targeted CENP-A chromatin at the LacO array was sufficient to direct the assembly of a functional centromere as indicated by the recruitment of the constitutive centromere-associated network proteins, the microtubule-binding protein NDC80, and the formation of stable kinetochore–microtubule attachments. An amino-terminal fragment of HJURP was able to assemble CENP-A nucleosomes in vitro, demonstrating that HJURP is a chromatin assembly factor. Furthermore, HJURP recruitment to endogenous centromeres required the Mis18 complex. Together, these data suggest that the role of the Mis18 complex in CENP-A deposition is to recruit HJURP and that the CENP-A nucleosome assembly activity of HJURP is responsible for centromeric chromatin assembly to maintain the epigenetic mark.