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Influence of Sex and Genetic Variability on Expression of X-Linked Genes In CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Genomics 98 (2011) 320–326 Contents lists available at ScienceDirect Genomics journal homepage: www.elsevier.com/locate/ygeno Influence of sex and genetic variability on expression of X-linked genes in human monocytes Raphaële Castagné a, Tanja Zeller b,1, Maxime Rotival a, Silke Szymczak c, Vinh Truong a, Arne Schillert c, David-Alexandre Trégouët a, Thomas Münzel b, Andreas Ziegler c, François Cambien a, Stefan Blankenberg b,1, Laurence Tiret a,⁎ a INSERM UMRS 937, Pierre and Marie Curie University (UPMC, Paris 6) and Medical School, 91 Bd de l'Hôpital 75013, Paris, France b II. Medizinische Klinik und Poliklinik, Johannes-Gutenberg Universität Mainz, Universitätsmedizin, Langenbeckstraße 1, 55131 Mainz, Germany c Institut für Medizinische Biometrie und Statistik, Universität zu Lübeck, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany article info abstract Article history: In humans, the fraction of X-linked genes with higher expression in females has been estimated to be 5% from Received 12 April 2011 microarray studies, a proportion lower than the 25% of genes thought to escape X inactivation. We analyzed Accepted 28 June 2011 715 X-linked transcripts in circulating monocytes from 1,467 subjects and found an excess of female-biased Available online 6 July 2011 − transcripts on the X compared to autosomes (9.4% vs 5.5%, pb2×10 5). Among the genes not previously known to escape inactivation, the most significant one was EFHC2 whose 20% of variability was explained by Keywords: sex. We also investigated cis expression quantitative trait loci (eQTLs) by analyzing 15,703 X-linked SNPs. The X chromosome inactivation Gene expression frequency and magnitude of X-linked cis eQTLs were quite similar in males and females. Few genes exhibited a Expression Quantitative Trait Locus stronger genetic effect in females than in males (ARSD, DCX, POLA1 and ITM2A). These genes would deserve Gender further investigation since they may contribute to sex pathophysiological differences. © 2011 Elsevier Inc. All rights reserved. 1. Introduction The exact mechanisms regulating expression status of genes on the inactive X are not fully elucidated. It is known that genes escaping In female mammals, one of the two X chromosomes is inactivated inactivation are not uniformly distributed along the X chromosome to maintain equal expression between sexes by a process called X but preferentially lie in the evolutionary younger strata of the X chromosome inactivation (XCI) [1]. XCI is a complex and tightly chromosome [5,6]. It has been hypothesized that long interspersed regulated mechanism governed by the XIST gene which encodes a elements (LINE-1), which are particularly abundant on the X non-coding functional RNA coating the chromosome from which it is chromosome and are enriched in the vicinity of inactivated genes, expressed and triggers inactivation [2]. Although most genes are might control spreading of XCI [7,8]. silenced by this process, it has long been recognized that some genes Although in vitro hybrid systems have greatly contributed to our escape XCI and are expressed from both the active X (Xa) and inactive knowledge of XCI, they do not reflect the in vivo processes. In X(Xi) chromosomes. Studies using rodent-human hybrid cell lines particular, these experiments do not quantify the difference of retaining an inactive human X chromosome have estimated that expressionbetweenmalesandfemalesforgenesthatescape about 25% of X-linked genes escape inactivation to some degree [3–5]. inactivation. These genes are of primary interest since they are potential contributors to variability between sexes for phenotypic and clinical traits. Recent studies have taken benefit from the microarray Abbreviations: XCI, X Chromosome Inactivation; LCL, Lymphoblastoid Cell Line; technology to compare expression profiles of the X chromosome eQTL, Expression Quantitative Trait Locus; SNP, Single Nucleotide Polymorphism. between males and females [9–12]. The largest study, based on ⁎ Correspondingauthorat:INSERMUMRS937,FacultédeMédecinePitié- lymphoblastoid cell lines (LCLs) from 210 individuals from the four Salpêtrière, 91 bd de l'Hôpital, 75634 PARIS cedex 13, France. Fax: +33 1 40 77 97 28. HapMap populations, reported 5% of X-linked genes showing higher E-mail addresses: [email protected] (R. Castagné), [email protected] (T. Zeller), [email protected] (M. Rotival), [email protected] expression in females than in males [9], a proportion much lower than (S. Szymczak), [email protected] (V. Truong), [email protected] the 25% of genes previously thought to escape XCI [5,6]. This (A. Schillert), [email protected] (D.-A. Trégouët), [email protected] underestimation might be partly explained by an insufficient power (T. Münzel), [email protected] (A. Ziegler), [email protected] to detect small sex differences, since expression from the inactive (F. Cambien), [email protected] (S. Blankenberg), [email protected] (L. Tiret). chromosome may be low or present only in a fraction of females [5]. 1 Present address: Department of General and Interventional Cardiology, University Heart Center, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Here, we report the results of a large-scale study including 1467 Hamburg, Germany. population-based subjects in whom the expression profile from 0888-7543/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2011.06.009 R. Castagné et al. / Genomics 98 (2011) 320–326 321 circulating monocytes was assessed [13].Wefirst examined to what 2.2. Expression level of X-linked transcripts according to sex extent the mechanism of XCI was supported by this dataset including 715 X-linked transcripts. Secondly, because the impact of X-linked For each of the 715 X-linked transcripts, the median and the genetic variability on expression may have profound consequences on variance of expression levels in males were plotted against those in disease traits, as attested by numerous X-linked diseases, we females (Fig. 1). Most of the data points lay on the diagonal investigated whether cis expression quantitative loci (eQTLs) on the suggesting that XCI was globally achieved for the majority of X chromosome differed between sexes. genes. There were however a few points departing from the diagonal, most of them showing a higher expression level in femalesthaninmales. 2. Results We then focused on the transcripts that were differentially expressed between sexes using a Bonferroni-corrected significance The study was conducted in 717 women and 750 men of European threshold to account for the number of transcripts tested (7.0×10−5). origin aged 35 to 74 years that were recruited in the Gutenberg Heart We distinguished transcripts that were expressed at a higher level in Study (GHS), a community-based project conducted in a single centre females (referred to as female-biased) from those that were in Germany [13]. Genome-wide expression profiles of peripheral expressed at a higher level in males (male-biased) [14]. We observed blood monocytes were generated using Illumina HT-12 BeadChip a higher proportion of female-biased than male-biased X-linked expression arrays and genome-wide genotyping was performed using transcripts (9.37% vs 5.59%, p=0.006). We tested whether these the Affymetrix Genome-Wide Human SNP Array 6.0. proportions were different from those observed on autosomes by resampling 106 sets of 715 autosomal transcripts. There was a significant excess of female-biased transcripts on the X chromosome 2.1. X-linked transcripts detected in human monocytes compared to autosomes (9.37% vs 5.46%, pb2×10−5) whereas the difference was not significant for male-biased transcripts (5.59% vs After removing probes with a bad quality score, 1156 probes were 4.65%, p=0.10). The higher proportion of female-biased genes is in unambiguously assigned to the X chromosome, corresponding to 706 X- agreement with the fact that a fraction of X-linked genes are known or linked genes. Analyses were performed at the probe level and, for suspected to escape from silencing [5,9–12]. simplicity, the term of “transcript” was used to denote a unique probe- Because the method of monocyte enrichment used in the present hybridization product (although in few cases the same transcript could study did not yield a 100% purity and even modest heterogeneity of be targeted by several probes). We focused on transcripts whose cell content may induce artefactual associations, we checked whether expression was detected, i.e. higher than the background signal, in≥5% contamination by non-monocyte cells might affect the differences of female samples or≥5% of male samples. These represented 715 X- observed. To estimate the relative amount of RNA coming from the linked (61.8%) transcripts corresponding to 503 unique genes (Supple- different blood cell types, we applied a deconvolution algorithm mentary Table 1). The majority of X-linked transcripts were detected proposed in [15] and used cell-specific expression profiles reported in with similar detection rates in males and females (Supplementary Fig. the HaemAtlas [16] to generate surrogate variables of contamination 1). XIST (X (inactive)-specifictranscript),thegeneresponsiblefor (see Methods). Adjusting for these variables hardly modified the inactivation of one of the X copies in females, was expressed at low difference between female-biased and male-biased X-linked genes levels, yet higher than the background signal, in 62% of males. (9.51% vs 5.73%, p=0.007). Fig. 1. Comparison of expression levels of X-linked transcripts between males and females. (A) Median of expression. The pink line represents the situation where transcripts would completely escape XCI in females (log2Y=log2X+1). The only point departing from this line is XIST, the gene responsible for X inactivation of one chromosome in females. (B) Variance of expression.
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