Genetic Expression Analysis Research Articles

A new tool in C. elegans reveals changes in secretory protein metabolism in ire-1-deficient animals.

We recently showed that the ire-1/xbp-1 arm of the UPR plays a crucial role in maintaining basic endoplasmic reticulum (ER) functions required for the metabolism of secreted proteins even during unstressed growth conditions. During these studies we realized that although C. elegans is a powerful system to study the genetics of many cellular processes; it lacks effective tools for tracking the metabolism of secreted proteins at the cell and organism levels. Here, we outline how genetic manipulations and expression analysis of a DAF-28::GFP translational fusion transgene can be combined to infer different steps in the life cycle of secretory proteins. We demonstrate how we have used this tool to reveal folding defects, clearance defects, and secretion defects in ire-1 and xbp-1 mutants. We believe that further studies using this tool will deepen the understanding of secretory protein metabolism.

Genetic variation within the Chrna7 gene modulates nicotine reward‐like phenotypes in mice

Mortality from tobacco smoking remains the leading cause of preventable death in the world, yet current cessation therapies are only modestly successful, suggesting new molecular targets are needed. Genetic analysis of gene expression and behavior identified Chrna7 as potentially modulating nicotine place conditioning in the BXD panel of inbred mice. We used gene targeting and pharmacological tools to confirm the role of Chrna7 in nicotine conditioned place preference (CPP). To identify molecular events downstream of Chrna7 that may modulate nicotine preference, we performed microarray analysis of α7 knock-out (KO) and wild-type (WT) nucleus accumbens (NAc) tissue, followed by confirmation with quantitative polymerase chain reaction (PCR) and immunoblotting. In the BXD panel, we found a putative cis expression quantitative trait loci (eQTL) for Chrna7 in NAc that correlated inversely to nicotine CPP. We observed that gain-of-function α7 mice did not display nicotine preference at any dose tested, whereas conversely, α7 KO mice demonstrated nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the α7 nicotinic acetylcholine receptor (nAChR)-selective agonist, PHA-543613, dose-dependently blocked nicotine CPP, which was restored using the α7 nAChR-selective antagonist, methyllycaconitine citrate (MLA). Our genomic studies implicated a messenger RNA (mRNA) co-expression network regulated by Chrna7 in NAc. Mice lacking Chrna7 demonstrate increased insulin signaling in the NAc, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation.

Association analysis of ERBB2 amplicon genetic polymorphisms and STARD3 expression with risk of gastric cancer in the Chinese population

The purpose of this study was to investigate whether risk of gastric cancer (GC) was associated with single nucleotide polymorphisms (SNPs) in a gene cluster on the chromosome 17q12-q21 (ERBB2 amplicon) in the Chinese Han population. We detected twenty-six SNPs in this gene cluster containing steroidogenic acute regulatory-related lipid transfer domain containing 3 (STARD3), protein phosphatase 1 regulatory subunit 1B (PPP1R1B/DARPP32), titin-cap (TCAP), per1-like domain containing 1(PERLD1/CAB2), human epidermal growth factor receptor-2 (ERBB2/HER2), zinc-finger protein subfamily 1A 3 (ZNFN1A3/IKZF3) and DNA topoisomerase 2-alpha (TOP2A) genes in 311 patients with GC and in 425 controls by Sequenom. We found no associations between genetic variations and GC risk. However, haplotype analysis implied that the haplotype CCCT of STARD3 (rs9972882, rs881844, rs11869286 and rs1877031) conferred a protective effect on the susceptibility to GC (P=0.043, odds ratio [OR]=0.805, 95% confidence intervals [95% CI]=0.643–0.992). The STARD3 rs1877031 TC genotype endued histogenesis of gastric mucinous adenocarcinoma and signet-ring cell carcinoma (P=0.021, OR=2.882, 95% CI=1.173–7.084). We examined the expression of STARD3 in 243 tumor tissues out of the 311 GC patients and 20 adjacent normal gastric tissues using immumohistochemical (IHC) analysis and tissue microarrays (TMA). The expression of STARD3 was observed in the gastric parietal cells and in gastric tumor tissues and significantly correlated with gender (P=0.004), alcohol drinking (P<0.001), tumor location (P=0.007), histological type (P=0.005) and differentiation (P=0.023) in GC. We concluded that the combined effect of haplotype CCCT of STARD3 might affect GC susceptibility. STARD3 expression might be related to the tumorigenesis of GC in the Chinese population.

Molecular pathology of acute kidney injury in a choline-deficient model and fish oil protective effect

The aim of this work was to investigate the potential protective effects of fish oil on the basis of kidney transcriptomic data on a nutritional experimental model. Male weanling Wistar rats were divided into four groups and fed choline-deficient (CD) and choline-supplemented (CS) diets with vegetable oil (VO) and menhaden oil (MO): CSVO, CDVO, CSMO and CDMO. Animals were killed after receiving the diets for 6 days. Total RNA was purified from the right kidney and hybridized to Affymetrix GeneChip Rat Gene 1.0 ST Array. Differentially expressed genes were analyzed. All CSVO, CSMO and CDMO rats showed no renal alterations, while all CDVO rats showed renal cortical necrosis. A thorough analysis of the differential expression between groups CSMO and CDMO was carried out. There were no differential genes for p < 0.01. The analysis of the differential expression between groups CSVO and CSMO revealed 32 genes, 11 were over-expressed and 21 were under-expressed in CSMO rats. This work was part of a large set of experiments and was used in a hypothesis-generating manner. The comprehensive analysis of genetic expression allowed confirming that menhaden oil has a protective effect on this nutritional experimental model and identifying 32 genes that could be responsible for that protection, including Gstp1. These results reveal that gene changes could play a role in renal injury.

Do post-translational beta cell protein modifications trigger type 1 diabetes?

Type 1 diabetes is considered an autoimmune disease characterised by specific T cell-mediated destruction of the insulin-producing beta cells. Yet, except for insulin, no beta cell-specific antigens have been discovered. This may imply that the autoantigens in type 1 diabetes exist in modified forms capable of specifically triggering beta cell destruction. In other immune-mediated diseases, autoantigens targeted by the immune system have undergone post-translational modification (PTM), thereby creating tissue-specific neo-epitopes. In a similar manner, PTM of beta cell proteins might create beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading to the T cell attack against beta cells is presented. In this model, PTM plays a prominent role in triggering beta cell destruction. We discuss literature of relevance and perform genetic and human islet gene expression analyses. Both direct and circumstantial support for the involvement of PTM in type 1 diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1 diabetes as a classical autoimmune disease is open for debate.

High-Throughput Genetic and Gene Expression Analysis of the RNAPII-CTD Reveals Unexpected Connections to SRB10/CDK8

The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is composed of heptapeptide repeats, which play a key regulatory role in gene expression. Using genetic interaction, chromatin immunoprecipitation followed by microarrays (ChIP-on-chip) and mRNA expression analysis, we found that truncating the CTD resulted in distinct changes to cellular function. Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels. The latter were largely mediated by promoter elements and in part were linked to the transcription factor Rpn4. The mediator subunit Cdk8 was enriched at promoters of these genes, and its removal not only restored normal mRNA and RNAPII occupancy levels, but also reduced the abnormally high cellular amounts of Rpn4. This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene. Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5–6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

Shox2 Regulates the Pacemaker Gene Program in Embryoid Bodies

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heartbeat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node can serve as a subsidiary pacemaker in cases of SAN failure or block. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of targeted therapies. Here we report temporal and spatial self-organized formation of the pacemaker and contracting tissues in three-dimensional aggregate cultures of mouse embryonic stem cells termed embryoid bodies (EBs). Using genetic marker expression and electrophysiological analyses we demonstrate that in EBs the pacemaker potential originates from a localized population of cells and propagates into the adjacent contracting region forming a functional syncytium. When Shox2, a major determinant of the SAN genetic pathway, was ablated we observed substantial slowing of spontaneous contraction rates and an altered gene expression pattern including downregulation of HCN4, Cx45, Tbx2, Tbx3, and bone morphogenetic protein 4 (BMP4); and upregulation of Cx40, Cx43, Nkx2.5, and Tbx5. This phenotype could be rescued by adding BMP4 to Shox2 knockout EBs in culture from days 6 to 16 of differentiation. When wild-type EBs were treated with Noggin, a potent BMP4 inhibitor, we observed a phenotype consistent with the Shox2 knockout EB. Altogether, we have generated a reproducible in vitro model that will be an invaluable tool for studying the molecular pathways regulating the development of cardiac pacemaker tissues.

Genetic, Physiological, and Gene Expression Analyses Reveal That Multiple QTL Enhance Yield of Rice Mega-Variety IR64 under Drought

BackgroundRice (Oryza sativa L.) is a highly drought sensitive crop, and most semi dwarf rice varieties suffer severe yield losses from reproductive stage drought stress. The genetic complexity of drought tolerance has deterred the identification of agronomically relevant quantitative trait loci (QTL) that can be deployed to improve rice yield under drought in rice. Convergent evidence from physiological characterization, genetic mapping, and multi-location field evaluation was used to address this challenge.Methodology/Principal FindingsTwo pairs of backcross inbred lines (BILs) from a cross between drought-tolerant donor Aday Sel and high-yielding but drought-susceptible rice variety IR64 were produced. From six BC4F3 mapping populations produced by crossing the +QTL BILs with the −QTL BILs and IR64, four major-effect QTL - one each on chromosomes 2, 4, 9, and 10 - were identified. Meta-analysis of transcriptome data from the +QTL/−QTL BILs identified differentially expressed genes (DEGs) significantly associated with QTL on chromosomes 2, 4, 9, and 10. Physiological characterization of BILs showed increased water uptake ability under drought. The enrichment of DEGs associated with root traits points to differential regulation of root development and function as contributing to drought tolerance in these BILs. BC4F3-derived lines with the QTL conferred yield advantages of 528 to 1875 kg ha−1 over IR64 under reproductive-stage drought stress in the targeted ecosystems of South Asia.Conclusions/SignificanceGiven the importance of rice in daily food consumption and the popularity of IR64, the BC4F3 lines with multiple QTL could provide higher livelihood security to farmers in drought-prone environments. Candidate genes were shortlisted for further characterization to confirm their role in drought tolerance. Differential yield advantages of different combinations of the four QTL reported here indicate that future research should include optimizing QTL combinations in different genetic backgrounds to maximize yield advantage under drought.

Two Related Pyrrolidinedione Synthetase Loci in Fusarium heterosporum ATCC 74349 Produce Divergent Metabolites

Equisetin synthetase (EqiS), from the filamentous fungus Fusarium heterosporum ATCC 74349, was initially assigned on the basis of genetic knockout and expression analysis. Increasing inconsistencies in experimental results led us to question this assignment. Here, we sequenced the F. heterosporum genome, revealing two hybrid polyketide-peptide proteins that were candidates for the equisetin synthetase. The surrounding genes in both clusters had the needed auxiliary genes that might be responsible for producing equisetin. Genetic mutation, biochemical analysis, and recombinant expression in the fungus enabled us to show that the initially assigned EqiS does not produce equisetin but instead produces a related 2,4-pyrrolidinedione, fusaridione A, that was previously unknown. Fusaridione A is methylated in the 3-position of the pyrrolidinedione, which has not otherwise been found in natural products, leading to spontaneous reverse-Dieckmann reactions. A newly described gene cluster, eqx, is responsible for producing equisetin.

The sld Genetic Defect: TWO INTRONIC CA REPEATS PROMOTE INSERTION OF THE SUBSEQUENT INTRON AND mRNA DECAY

The autosomal recessive mutation, sld, attenuates mucous cell expression in murine sublingual glands with corresponding effects on mucin 19 (Muc19). We conducted a systematic study including genetic mapping, sequencing, and functional analyses to elucidate a mutation to explain the sld phenotype in neonatal mice. Genetic mapping and gene expression analyses localized the sld mutation within the gene Muc19/Smgc, specifically attenuating Muc19 transcripts, and Muc19 knock-out mice mimic the sld phenotype in neonates. Muc19 transcription is unaffected in sld mice, whereas mRNA stability is markedly decreased. Decreased mRNA stability is not due to a defect in 3'-end processing nor to sequence differences in Muc19 transcripts. Comparative sequencing of the Muc19/Smgc gene identified four candidate intronic mutations within the Muc19 coding region. Minigene splicing assays revealed a novel splicing event in which insertion of two additional repeats within a CA repeat region of intron 53 of the sld genome enhances retention of intron 54, decreasing the levels of correctly spliced transcripts. Moreover, pateamine A, an inhibitor of nonsense-mediated mRNA decay, inhibits degradation of aberrant Muc19 transcripts. The mutation in intron 53 thus enhances aberrant splicing leading to degradation of aberrant transcripts and decreased Muc19 message stability, consistent with the sld phenotype. We propose a working model of the unique splicing event enhanced by the mutation, as well as putative explanations for the gradual but limited increase in Muc19 glycoprotein expression and its restricted localization to subpopulations of mucous cells in sld mice during postnatal gland development.

BRANCHED1 Interacts with FLOWERING LOCUS T to Repress the Floral Transition of the Axillary Meristems inArabidopsis

Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.

A synonymous mutation in NOD2 gene was significantly associated with non-specific digestive disorder in rabbit

Nucleotide-binding oligomerization domain containing 2 (NOD2) plays a pivotal role in the host innate and adaptive immunity by recognizing the pathogenic agents. Therefore, its genetic polymorphisms and association with susceptibility to infectious diseases have been widely reported in human and farm animals. In the present study, we investigated the genetic polymorphisms in 3171bp coding region of NOD2 gene and association with non-specific digestive disorder (NSDD) in rabbit. A total of four coding single-nucleotide polymorphisms (cSNPs) were detected. Among them, c.2961C>T was further genotyped for case (n=176) and control (n=130) based on association analysis, which revealed that C allele carried the potential protective role for susceptibility to NSDD with the odds ratio (OR) values of 0.52 (95% confidence interval (CI) 0.37–0.73, P<0.01). Under the dominant inheritance model, CC genotype was associated with decreased susceptibility to NSDD (OR=0.38, 95% CI 0.24–0.60, P<0.01). Along with the aggravation of NSDD, we observed higher mRNA expression of NOD2 gene (P<0.05). However, the mRNA expression pattern of CC genotype would be interacted by the different status of NSDD, which only showed the significantly increased level in severe NSDD group (P<0.05). These results revealed by genetic association and gene expression analysis suggested that the NOD2 gene was associated with the susceptibility to NSDD in rabbit. However, the causative mutations linked to c.2961C>T and corresponding functional depiction should be further explored by performing exhaustive genetic studies.

Laser capture microdissection of metachromatically stained skeletal muscle allows quantification of fiber type specific gene expression

Skeletal muscle contains various myofiber types closely associated with satellite stem cells, vasculature, and neurons, thus making it difficult to perform genetic or proteomic expression analysis with sufficient cellular specificity to resolve differences at the individual cell or myofiber type level. Here, we describe the combination of a simple histochemical method capable of simultaneously identifying Type I, IIA, IIB, and IIC myofibers followed by laser capture micro-dissection (LCM) to compare the expression profiles of individual fiber types, myonuclear domains, and satellite cells in frozen muscle sections of control and atrophied muscle. Quantitative RT-PCR (qPCR) was used to verify the integrity of the cell-specific RNAs harvested after histologic staining, while qPCR for specific genes of interest was used to quantify atrophy-associated changes in mRNA. Our data demonstrate that the differential myofiber atrophy previously described by histologic means is related to differential expression of atrophy-related genes, such as MuRF1 and MAFbx (a.k.a. Atrogin-1), within different myofiber type populations. This spatially resolved molecular pathology (SRMP) technique allowed quantitation of atrophy-related gene products within individual fiber types that could not be resolved by expression analysis of the whole muscle. The present study demonstrates the importance of fiber type specific expression profiling in understanding skeletal muscle biology especially during muscle atrophy and provides a practical method of performing such research.

The Metabolomic Signature of Malignant Glioma Reflects Accelerated Anabolic Metabolism

Although considerable progress has been made toward understanding glioblastoma biology through large-scale genetic and protein expression analyses, little is known about the underlying metabolic alterations promoting their aggressive phenotype. We conducted global metabolomic profiling on patient-derived glioma specimens and identified specific metabolic programs differentiating low- and high-grade tumors, with the metabolic signature of glioblastoma reflecting accelerated anabolic metabolism. When coupled with transcriptional profiles, we identified the metabolic phenotype of the mesenchymal subtype to consist of accumulation of the glycolytic intermediate phosphoenolpyruvate and decreased pyruvate kinase activity. Unbiased hierarchical clustering of metabolomic profiles identified three subclasses, which we term energetic, anabolic, and phospholipid catabolism with prognostic relevance. These studies represent the first global metabolomic profiling of glioma, offering a previously undescribed window into their metabolic heterogeneity, and provide the requisite framework for strategies designed to target metabolism in this rapidly fatal malignancy.

Abstract 131: Differential Regulation of Pitx2 Expression in Pulmonary Vein Muscle Sleeves and Left Atrium with Aging: Implication for Pulmonary Vein Triggers

Introduction: Pitx2, Paired-like homeodomain transcription factor 2, is a homeobox transcription factor that regulates left-right asymmetry during cardiogenesis. Pitx2 insufficiency in the left atrium (LA) has been linked to LA remodeling and arrhythmogenesis in age-related atrial fibrillation (AF). However, the main triggers for AF reside in pulmonary vein muscle sleeves (PVs). Whether Pitx2 insufficiency in PVs also plays a pathogenic role in generating trigger foci remains to be determined. Also, changes in Pitx2 expression in PVs with age remain unclear. We use qRT-PCR and Illumina microarrays to determine age-related changes in Pitx2 expression of PVs in wild type (WT) &amp; heterozygous Pitx2cnull+/- mice, and in humans with or without AF. Methods: PV and LA regions were obtained from 1-23 month-old WT and Pitx2cnull+/- mice. Human PVs were obtained from ~110D human fetal hearts (E110D aborted fetus), two groups of adult donor hearts [20-30s year-old (yo) and 40-50 yo] and two patients with persistent AF. These tissues were used for genetic expression analysis. Results: Pitx2c expression decreases slightly with age in mouse LA and PV tissues. The expression levels of Pitx2c of Pitx2cnull+/- mice in LA are consistently at 55.2±10.5% of WT but at near-equal levels in PVs over four different age groups. In contrast, during human development, Pitx2 expression in PV muscle sleeves first decreases from ~E110D to ~30 yo but increases from 30 to 50 yo. In patients with AF, Pitx2 expression in PVs is only lower by 15% while HCN4 expression is higher by 13.5% when compared to control PVs at &gt;50 yo. Expressions of type 2 Ryanodine receptors and calsequestrin in human PVs increase with age but decrease by ~30% with AF, suggesting impaired calcium handling abilities may play more significant roles than Pitx2 in the generation of PV triggers of AF. Conclusions: Genetic analysis of Pitx2c expression in PV muscle sleeves and LA tissues of WT and Pitx2cnull+/- mice revealed differential expression patterns. In human PVs, Pitx2 expression shows a regulation pattern with age distinct from that in mice, and genes linked to intracellular calcium handling may play a more important role than Pitx2 in generating trigger foci in PVs to initiate AF.

Analysis of genetic expression in the soft palate of patients with obstructive sleep apnea

Conclusion: Genetic dysregulation may be associated with development of obstructive sleep apnea (OSA). Objectives: Genetic expression was investigated in the soft palate of patients with OSA to evaluate the biomolecular characteristics of the tissue where airway obstruction actually occurs. Methods: Soft palate mucosa was obtained during uvulopalatal flap surgery. Three patients with apnea/hypopnea index (AHI) >30/h and three simple snoring patients with AHI < 5/h were enrolled. After total RNA was extracted and amplified, gene expression levels were determined by microarray analysis, and relative signal intensities for each gene were evaluated. Results: Of the 45,034 genes analyzed, 137 were up-regulated in OSA patients compared with simple snorers and 95 were down-regulated. Gene network analysis showed that genes related to RNA interference and transcription were most commonly involved in the functional pathophysiology of OSA. In particular, genes encoding proliferating cell nuclear antigen and proteasome 26S subunit ATPase 6 were functionally connected with other genes.

Effect of normalization on statistical and biological interpretation of gene expression profiles.

An under-appreciated aspect of the genetic analysis of gene expression is the impact of post-probe level normalization on biological inference. Here we contrast nine different methods for normalization of an Illumina bead-array gene expression profiling dataset consisting of peripheral blood samples from 189 individual participants in the Center for Health Discovery and Well Being study in Atlanta, quantifying differences in the inference of global variance components and covariance of gene expression, as well as the detection of variants that affect transcript abundance (eSNPs). The normalization strategies, all relative to raw log2 measures, include simple mean centering, two modes of transcript-level linear adjustment for technical factors, and for differential immune cell counts, variance normalization by interquartile range and by quantile, fitting the first 16 Principal Components, and supervised normalization using the SNM procedure with adjustment for cell counts. Robustness of genetic associations as a consequence of Pearson and Spearman rank correlation is also reported for each method, and it is shown that the normalization strategy has a far greater impact than correlation method. We describe similarities among methods, discuss the impact on biological interpretation, and make recommendations regarding appropriate strategies.

Low-density lipoprotein receptor-related protein 2 gene is associated with egg-quality traits in dwarf layers

Some members of the low-density lipoprotein receptor (LDLR) family play important roles in the regulation of lipoprotein metabolism and egg quality traits. Low-density lipoprotein receptor-related protein 2 (LRP2) gene belongs to the LDLR super family, and widely expresses in many tissues. This work identified and genotyped 1 single-nucleotide polymorphism (SNP), T14347C, at 3'-UTR of the LRP2 using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and analyzed the effects of the SNP (T14347C) on egg-quality traits in 544 dwarf hens from 44 sire families. Frequencies of this SNP in the studied population did not agree with the Hardy-Weinberg equilibrium (P < 0.0001). Egg weight, albumen weight, albumen height, and albumen ratio of the TT genotype were significantly higher than those of the CC genotype (P < 0.05), whereas eggshell ratio of the TT genotype was significantly lower than that of the CC genotype (P < 0.05). The relative expression level of the LRP2 gene in the magnum was determined by real-time quantitative PCR. The gene expression of genotype CC individuals was significantly higher than that of TT and CT birds (P < 0.05). By combining both genetic effects and expression analyses results, we propose that the LRP2 gene is a good candidate gene, exhibiting a key role in albumen formation processes.

TG20, a Transgenically-Derived Anti-CD20 Monoclonal Antibody, Exhibits Enhanced Cytotoxicity Against Cells with Low Levels of CD20

Abstract 2730CD20 is a cell-surface glycoprotein that is highly expressed on most B-cells, tightly restricted to the B-cell lineage, and not expressed on either precursor lymphoid cells or the majority of plasma cells. These characteristics make CD20 an appealing target for mAb therapy of B-cell malignancies and B-cell dependent autoimmune conditions, because antibody production is maintained during therapy and B-cell regeneration post-CD20 mAb treatment is facilitated. Among several marketed anti-CD20 mAbs, rituximab is the prototype; a chimeric human–mouse type-1 antibody that has proven efficacy in a wide variety of mature B-cell malignancies, including CD20+ B-cell lymphoma, and has also been approved for the treatment of refractory rheumatoid arthritis. However, patients do not always respond to this therapy, and it has been reported that close to 60% of follicular lymphoma patients previously treated with chemotherapy, while initially responsive, become resistant upon repeat treatment with rituximab monotherapy. Furthermore, the high cost of treatment with rituximab and other anti-CD20 mAbs severely curtails their availability to patients in emerging economies, as well as their use post treatment for maintenance therapy.Transgenic production offers an easily scalable system for the cost-effective manufacturing of large amounts of complex therapeutic proteins. The regulatory approvals of ATryn® (recombinant antithrombin), first by the EMA (August 2006) and by the FDA (February 2009) have provided a strong regulatory validation of this production platform. Produced in the milk of transgenic goats, TG20, a novel chimeric IgG1 strongly binding a specific discontinuous epitope on CD20 (KD = 1–2 × 10−8 M), is being developed as a second-generation anti-CD20 mAb with enhanced cytotoxic activity. Using regulatory sequences from the caprine beta-casein gene to target expression of the TG20 heavy- and light-chains to the lactating mammary gland, 7 independent lines of transgenic goats were generated by somatic cell nuclear transfer. Induced lactation studies indicated that these lines expressed TG20 in their milk at levels varying from 2 g/liter up to ∼ 10 g/liter. Following genetic characterization (Southern blotting, FISH analysis) and expression analysis (protein and peptide mapping, glycosylation profiling, amino-acid sequence analysis), one line was selected for expansion and further development.In-vitro studies comparing TG20 to rituximab showed a significantly increased ability to activate effector cells expressing the FcgRIIIa receptor (CD16a). Not surprisingly, this translates to a significantly increased ability to activate NK cells and to induce target lysis. This increased ADCC has been demonstrated both with CD20+ Raji cells and with primary B-cells isolated from patients with B-CLL (TG20 EC50 = 3.6 ng/ml vs. rituximab EC50 = 59.4 ng/ml). The ability of TG20 to activate the complement system was assessed using human serum as complement and WIL2-S cells as target exhibiting a > 50% increase in CDC activity vs. rituximab (TG20 = 170% CDC activity vs. rituximab = 100%). Finally, studies conducted in Cynomolgus monkeys were performed to assess pharmacokinetics and in-vivo depletion activities of TG20.In conclusion, TG20 is a highly active anti-CD20 mAb showing interesting characteristics in terms of cytotoxicity that makes it a promising agent for indications in which rituximab is poorly active. Furthermore, the use of the cost-efficient transgenic production platform for the large-scale production of this antibody drug candidate may allow expanded access of anti-CD20 therapy to those who currently cannot afford these expensive treatments, especially in emerging market countries. Disclosures:Echelard:GTC Biotherapeutics, Inc.: Employment. Pollock:GTC Biotherapeutics, Inc.: Employment. De Coupade:LFB Biotechnologies: Employment. Groseil Olivier:LFB Biotechnologies: Employment. Brune:LFB Biotechnologies: Employment. Chen:GTC Biotherapeutics, Inc.: Employment. Masiello:GTC Biotherapeutics, Inc.: Employment. Williams:GTC Biotherapeutics, Inc.: Employment. Gavin:GTC Biotherapeutics, Inc.: Employment. Chtourou:LFB Biotechnologies: Employment. Meade:GTC Biotherapeutics, Inc.: Employment.