Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer

Background

We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.

Methodology/Principal Findings

We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohisotchemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10⁻⁴ when the SNP was examined alone).

Conclusions/Significance

The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.     

The Proneural Molecular Signature Is Enriched in Oligodendrogliomas and Predicts Improved Survival among Diffuse Gliomas

The Cancer Genome Atlas Project (TCGA) has produced an extensive collection of ‘-omic’ data on glioblastoma (GBM), resulting in several key insights on expression signatures. Despite the richness of TCGA GBM data, the absence of lower grade gliomas in this data set prevents analysis genes related to progression and the uncovering of predictive signatures. A complementary dataset exists in the form of the NCI Repository for Molecular Brain Neoplasia Data (Rembrandt), which contains molecular and clinical data for diffuse gliomas across the full spectrum of histologic class and grade. Here we present an investigation of the significance of the TCGA consortium''s expression classification when applied to Rembrandt gliomas. We demonstrate that the proneural signature predicts improved clinical outcome among 176 Rembrandt gliomas that includes all histologies and grades, including GBMs (log rank test p = 1.16e-6), but also among 75 grade II and grade III samples (p = 2.65e-4). This gene expression signature was enriched in tumors with oligodendroglioma histology and also predicted improved survival in this tumor type (n = 43, p = 1.25e-4). Thus, expression signatures identified in the TCGA analysis of GBMs also have intrinsic prognostic value for lower grade oligodendrogliomas, and likely represent important differences in tumor biology with implications for treatment and therapy. Integrated DNA and RNA analysis of low-grade and high-grade proneural gliomas identified increased expression and gene amplification of several genes including GLIS3, TGFB2, TNC, AURKA, and VEGFA in proneural GBMs, with corresponding loss of DLL3 and HEY2. Pathway analysis highlights the importance of the Notch and Hedgehog pathways in the proneural subtype. This demonstrates that the expression signatures identified in the TCGA analysis of GBMs also have intrinsic prognostic value for low-grade oligodendrogliomas, and likely represent important differences in tumor biology with implications for treatment and therapy.     

An efficient paradigm for genetic epidemiology cohort creation

Development of novel methodologies to efficiently create large genetic epidemiology cohorts is needed. Here we describe a rapid, precise and cost-efficient method for collection of DNA from cases previously experiencing an osteoporotic fracture by identifying cases using and administrative health-care databases. Over the course of 14 months we collected DNA from 1,130 women experiencing an osteoporotic fracture, at a cost of $54 per sample. This cohort is among the larger DNA osteoporotic fracture collections in the world. The novel method described addresses a major unmet health care research need and is widely applicable to any disease that can be identified accurately through administrative data.     

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.Download video file.^{(159M, mp4)}     

A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VeGF and pDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments.The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting pDGFRβ and VeGF-A were selected for superior stability. the scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. assembly of a bsAb using stable monomeric units allowed development of an anti-pDGFRB/VeGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.Key words: bispecific, antibody, PDGFRβ, VEGF-A, stability, angiogenesis     

Toxoplasma IgG and IgA,but not IgM,antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites in the brain during the chronic stage of infection

Toxoplasma IgG and IgA, but not IgM, antibody titers were significantly higher in immunocompetent mice with cerebral proliferation of tachyzoites during the chronic stage of infection than those treated with sulfadiazine to inhibit the parasite growth. Their IgG and IgA antibody titers correlated significantly with the amounts of tachyzoite-specific SAG1 mRNA in their brains. In contrast, neither IgG, IgA, nor IgM antibody titers increased following two different doses of challenge infection in chronically infected mice. Increased antibody titers in IgG and IgA but not IgM may be a useful indicator suggesting an occurrence of cerebral tachyzoite growth in immunocompetent individuals chronically infected with Toxoplasma gondii.     

The joint evolution of mating system and pollen performance: Predictions regarding male gametophytic evolution in selfers vs. outcrossers

Studies of sexual selection in plants historically have focused on pollinator attraction, pollen transfer, gametophytic competition, and post-fertilization discrimination by maternal plants. Pollen performance (the speeds of germination and pollen tube growth) in particular is thought to be strongly subject to intrasexual selection, but the effect of mating system on this process has not been rigorously evaluated. Here we propose four predictions derived from the logic that pollen performance should evolve with mating system as an adaptive response to: (1) the competitive environment among pollen genotypes and (2) variation among female genotypes regularly encountered by a given pollen genotype. First, as previously predicted, due to the higher potential for intense selection among diverse pollen genotypes in outcrossing relative to selfing taxa, pollen should evolve to germinate and/or to grow more rapidly in outcrossers than in selfers. Second, due to stronger selection on pollen performance in outcrossing than in selfing taxa, heritable variation in pollen tube growth rate is more likely to be purged in outcrossers. In selfers, by contrast, genetic variation in pollen tube growth rates may readily accumulate because selfing reduces the number of genetically distinct male gametophytes likely to be deposited on any given stigma, thereby relaxing selection on male gametophytic traits. A summary of published studies presented here provides preliminary support for this prediction. Third, due to the high probability that the pollen of outcrossing individuals will be exposed to multiple pistil genotypes, we predict that the pollen of habitually outcrossing taxa will evolve to perform more consistently across female genotypes than the pollen of selfing taxa. Fourth, we predict that epistatic interactions between pollen and pistil genotypes are more likely to evolve in selfers than in outcrossers. We suggest several empirical approaches that may be used to test these predictions.     

A Locus on Chromosome 1p36 Is Associated with Thyrotropin and Thyroid Function as Identified by Genome-wide Association Study

Thyroid hormones are key regulators of cellular growth, development, and metabolism, and thyroid disorders are a common cause of ill health in the community. Circulating concentrations of thyrotropin (TSH), thyroxine (T4) and triiodothyronine (T3) have a strong heritable component and are thought to be under polygenic control, but the genes responsible are mostly unknown. In order to identify genetic loci associated with these metabolic phenotypes, we performed a genome-wide association study of 2,120,505 SNPs in 2014 female twins from the TwinsUK study and found a significant association between rs10917469 on chromosome 1p36.13 and serum TSH (p = 3.2 × 10⁻⁸). The association of rs10917469 with serum TSH was replicated (p = 2.0 × 10⁻⁴) in an independent community-based sample of 1154 participants in the Busselton Health Study. This SNP is located near CAPZB, which might be a regulator of TSH secretion and thus of pituitary-thyroid axis function. Twenty-nine percent of white individuals carry the variant, and the difference in mean TSH concentrations between wild-type individuals and those homozygous for the minor G allele was 0.5 mU/l, which is likely to be clinically relevant. We also provide evidence of suggestive association (p < 5.0 × 10⁻⁶) of other SNPs with serum TSH, free T4, and free T3 concentrations, and these SNPs might be good targets for further studies. These results advance understanding of the genetic basis of pituitary-thyroid axis function and metabolic regulation.     

A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.     

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.