Simulating pedigrees ascertained for multiple disease-affected relatives

Background

Studies that ascertain families containing multiple relatives affected by disease can be useful for identification of causal, rare variants from next-generation sequencing data.

Results

We present the R package SimRVPedigree, which allows researchers to simulate pedigrees ascertained on the basis of multiple, affected relatives. By incorporating the ascertainment process in the simulation, SimRVPedigree allows researchers to better understand the within-family patterns of relationship amongst affected individuals and ages of disease onset.

Conclusions

Through simulation, we show that affected members of a family segregating a rare disease variant tend to be more numerous and cluster in relationships more closely than those for sporadic disease. We also show that the family ascertainment process can lead to apparent anticipation in the age of onset. Finally, we use simulation to gain insight into the limit on the proportion of ascertained families segregating a causal variant. SimRVPedigree should be useful to investigators seeking insight into the family-based study design through simulation.

     

Effect of plant root symbionts on performance of native woody species in competition with an invasive grass in multispecies microcosms

The majority of terrestrial plants form mutualistic associations with arbuscular mycorrhizal fungi (AMF) and rhizobia (i.e., nitrogen‐fixing bacteria). Understanding these associations has important implications for ecological theory and for restoration practice. Here, we tested whether the presence of AMF and rhizobia influences the performance of native woody plants invaded by a non‐native grass in experimental microcosms. We planted eight plant species (i.e., Acacia acuminata, A. microbotrya, Eucalyptus loxophleba subsp. loxophleba, E. astringens, Calothamnus quadrifidus, Callistemon phoeniceus, Hakea lissocarpha and H. prostrata) in microcosms of field‐conditioned soil with and without addition of AMF and rhizobia in a fully factorial experimental design. After seedling establishment, we seeded half the microcosms with an invasive grass Bromus diandrus. We measured shoot and root biomass of native plants and Bromus, and on roots, the percentage colonization by AMF, number of rhizobia‐forming nodules and number of proteaceous root clusters. We found no effect of plant root symbionts or Bromus addition on performance of myrtaceous, and as predicted, proteaceous species as they rely little or not at all on AMF and rhizobia. Soil treatments with AMF and rhizobia had a strong positive effect (i.e., larger biomass) on native legumes (A. microbotrya and A. acuminata). However, the beneficial effect of root symbionts on legumes became negative (i.e., lower biomass and less nodules) if Bromus was present, especially for one legume, i.e., A. acuminata, suggesting a disruptive effect of the invader on the mutualism. We also found a stimulating effect of Bromus on root nodule production in A. microbotrya and AMF colonization in A. acuminata which could be indicative of legumes’ increased resource acquisition requirement, i.e., for nitrogen and phosphorus, respectively, in response to the Bromus addition. We have demonstrated the importance of measuring belowground effects because the aboveground effects gave limited indication of the effects occurring belowground.     

Significance of Liquid Biopsy for Monitoring and Therapy Decision of Colorectal Cancer

PURPOSE: Despite therapeutic improvements, all patients with nonresectable metastatic colorectal cancer (mCRC) acquire resistance to treatment probably due to the growth of mutated clones. In contrast to tissue-based studies, liquid biopsies have enabled the opportunity to reveal emerging resistance to treatment by detecting mutated clones and noninvasively monitoring clonal dynamics during therapy. METHODS: The courses of three patients with mCRC who were initially RAS wild-type were monitored longitudinally using liquid biopsy with long-term follow-up of up to 20 sequential samples. Detection of fragmented RAS mutated circulating cell-free DNA (cf)DNA in plasma was performed by BEAMing. In addition, plasma digital droplet PCR was used to detect and quantify BRAF and PIK3CA mutated cfDNA. Changes of mutational load were correlated with imaging data. RESULTS: A combination of liquid biopsy and radiological imaging enabled visualization of the occurrence of clonal redistribution after discontinuation of anti-EGFR mAb therapy, as well as emerging RAS mutations during therapy with anti-EGFR mAb indicating resistance. Furthermore, we found that growth of RAS mutated clones is independent of direct selective pressure by anti-EGFR therapy, which is a significant and new finding of this study. CONCLUSIONS: Our findings demonstrated the whole spectrum of clonal selection and redistribution of mutated cell clones leading to acquired resistance. Given our observation that the growth of RAS mutated clones can evolve even in the absence of anti-EGFR mAb therapy, there is a clear imperative to monitor RAS mutations in serial blood draws in all RAS wild-type patients in general and independent of the therapy.     

The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex's integrity and for cell viability in Saccharomyces cerevisiae.

Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.     

SALIVARY ANDROGEN-BINDING PROTEIN (ABP) MEDIATES SEXUAL ISOLATION IN MUS MUSCULUS

We wanted to determine whether the microevolution of the mouse salivary androgen-binding protein (ABP) Alpha subunit gene (Abpa) could mediate sexual selection and thereby have a potential role in maintaining gene pool integrity where radiating mouse subspecies make secondary contact. This hypothesis is based upon previous work in this laboratory, which has shown that each subspecies apparently has its own allele and that these alleles have a 25-fold excess of nonsynonymous/synonymous base substitutions compared to an average protein under purifying selection. We provide direct evidence for ABP-assortative mate selection in a laboratory setting: Mus musculus domesticus and M. m. musculus female mice recognize and discriminate between the territories of male mice that essentially differ solely in their Abpa genotype and, when the males are present, the female prefers to mate with the one of her own ABP type. The observation that females could differentiate between the territories of the two males when those mice were absent suggests that the males marked their territories with ABP. In this study, we also detected ABP on the pelts of male mice and in their environment. It is likely that the animals apply the protein to their pelts by licking and that it is then deposited in their surroundings. We suggest that females of the two subspecies are able to discriminate between males of those subspecies on the basis of this protein molecule. Mouse salivary ABP might present a worthwhile system with which to study a prezygotic isolation mechanism in a mammal.     

Human ex vivo carcinoma cells produce transforming growth factor β and thereby can inhibit lymphocyte functions in vitro

 We tested 20 human carcinoma samples for the production of transforming growth factor β (TGFβ) in vitro. Tumour cell suspensions without obvious contamination with non-malignant cells were kept in culture conditions for 16 h and their supernatants were added to CCL-64 cells. The proliferation of these cells is inhibited by TGFβ. According to this assay, the supernatants contained both active and latent TGFβ. In addition, the supernatants were found to suppress the spontaneous cytotoxic function and activation of T-cell-enriched lymphocyte populations. A specific monoclonal antibody (mAb) counteracted these effects and therefore we concluded that they were mediated to a large extent by TGFβ. In line with the results obtained with the supernatants, activation of lymphocytes could also be inhibited by tumour cells and their inhibitory effect was weaker in the presence of the TGFβ-specific mAb. It is important to note that, when TGFβ-specific mAb was added to autologous mixed lymphocyte/tumour cell cultures, lymphocyte activation occurred more often. These results thus substantiate the assumption that production of TGFβ may help the survival of potentially immunogenic tumour cells in immunocompetent patients. Received: 21 August 1996 / Accepted: 12 November 1996     

Three-dimensional Patterns and Redistribution of Myosin II and Actin in Mitotic Dictyostelium Cells

Myosin II is not essential for cytokinesis in cells of Dictyostelium discoideum that are anchored on a substrate (Neujahr, R., C. Heizer, and G. Gerisch. 1997. J. Cell Sci. 110:123–137), in contrast to its importance for cell division in suspension (DeLozanne, A., and J.A. Spudich. 1987. Science. 236:1086–1091; Knecht, D.A., and W.F. Loomis. 1987. Science. 236: 1081–1085.). These differences have prompted us to investigate the three-dimensional distribution of myosin II in cells dividing under one of three conditions: (a) in shaken suspension, (b) in a fluid layer on a solid substrate surface, and (c) under mechanical stress applied by compressing the cells. Under the first and second conditions outlined above, myosin II does not form patterns that suggest a contractile ring is established in the furrow. Most of the myosin II is concentrated in the regions that flank the furrow on both sides towards the poles of the dividing cell. It is only when cells are compressed that myosin II extensively accumulates in the cleavage furrow, as has been previously described (Fukui, Y., T.J. Lynch, H. Brzeska, and E.D. Korn. 1989. Nature. 341:328–331), i.e., this massive accumulation is a response to the mechanical stress. Evidence is provided that the stress-associated translocation of myosin II to the cell cortex is a result of the dephosphorylation of its heavy chains. F-actin is localized in the dividing cells in a distinctly different pattern from that of myosin II. The F-actin is shown to accumulate primarily in protrusions at the two poles that ultimately form the leading edges of the daughter cells. This distribution changes dynamically as visualized in living cells with a green fluorescent protein–actin fusion.