Accounting for population stratification in practice: a comparison of the main strategies dedicated to genome-wide association studies

Genome-Wide Association Studies are powerful tools to detect genetic variants associated with diseases. Their results have, however, been questioned, in part because of the bias induced by population stratification. This is a consequence of systematic differences in allele frequencies due to the difference in sample ancestries that can lead to both false positive or false negative findings. Many strategies are available to account for stratification but their performances differ, for instance according to the type of population structure, the disease susceptibility locus minor allele frequency, the degree of sampling imbalanced, or the sample size. We focus on the type of population structure and propose a comparison of the most commonly used methods to deal with stratification that are the Genomic Control, Principal Component based methods such as implemented in Eigenstrat, adjusted Regressions and Meta-Analyses strategies. Our assessment of the methods is based on a large simulation study, involving several scenarios corresponding to many types of population structures. We focused on both false positive rate and power to determine which methods perform the best. Our analysis showed that if there is no population structure, none of the tests led to a bias nor decreased the power except for the Meta-Analyses. When the population is stratified, adjusted Logistic Regressions and Eigenstrat are the best solutions to account for stratification even though only the Logistic Regressions are able to constantly maintain correct false positive rates. This study provides more details about these methods. Their advantages and limitations in different stratification scenarios are highlighted in order to propose practical guidelines to account for population stratification in Genome-Wide Association Studies.     

Perforin rapidly induces plasma membrane phospholipid flip-flop

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.     

Shedding light on the chemical diversity of ectopic calcifications in kidney tissues: diagnostic and research aspects

In most industrialized countries, different epidemiologic studies show that chronic renal failure is dramatically increasing. Such major public health problem is a consequence of acquired systemic diseases such as type II diabetes, which is now the first cause for end stage renal failure. Furthermore, lithogenic diseases may also induce intratubular crystallization, which may finally result in end-stage renal failure (ESRF). Up to now, such rare diseases are often misdiagnosed. In this study, based on twenty four biopsies, we show that SR μFTIR (Synchrotron Radiation-μFourier transform infrared) spectroscopy constitutes a significant opportunity to characterize such pathological μcalcifications giving not only their chemical composition but also their spatial distribution in the tissues. This experimental approach offers new opportunities to the clinicians to describe at the cell level the physico-chemical processes leading to the formation of the pathological calcifications which lead to ESRF.     

Identification of the chemokine CX3CL1 as a new regulator of malignant cell proliferation in epithelial ovarian cancer

Background

Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX₃CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX₃CL1 in EOC.

Results

Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX₃CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX₃CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX₃CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX₃CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX₃CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX₃CL1 products. Conversely, CX₃CL1 promoted through its binding to CX₃CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX₃CL1 and faster tumor growth.

Conclusion

Our findings highlight the previously unappreciated constitutive expression of CX₃CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients.     

Larger testes are associated with a higher level of polyandry, but a smaller ejaculate volume, across bushcricket species (Tettigoniidae)

While early models of ejaculate allocation predicted that both relative testes and ejaculate size should increase with sperm competition intensity across species, recent models predict that ejaculate size may actually decrease as testes size and sperm competition intensity increase, owing to the confounding effect of potential male mating rate. A recent study demonstrated that ejaculate volume decreased in relation to increased polyandry across bushcricket species, but testes mass was not measured. Here, we recorded testis mass for 21 bushcricket species, while ejaculate (ampulla) mass, nuptial gift mass, sperm number and polyandry data were largely obtained from the literature. Using phylogenetic-comparative analyses, we found that testis mass increased with the degree of polyandry, but decreased with increasing ejaculate mass. We found no significant relationship between testis mass and either sperm number or nuptial gift mass. While these results are consistent with recent models of ejaculate allocation, they could alternatively be driven by substances in the ejaculate that affect the degree of polyandry and/or by a trade-off between resources spent on testes mass versus non-sperm components of the ejaculate.     

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.