Common SNPs within or near Three Immune Response Genes Implicated in the Risk of FVIII Immunogenicity in Hemophilia A Do Not Influence Steady-State Levels of Their Encoded mRNAs

Abstract Abstract 3366 Background/Aims: The hemophilia A (HA)-causing Factor (F)VIII gene (F8) mutation type is a well-established determinant of risk for the development of alloimmune inhibitors that neutralize replacement FVIII proteins in ∼20% of all HA patients. Studies have investigated variants of immune response genes to determine if they may account for the inter-individual variability in FVIII immunogenicity observed in patients with the same F8 abnormality, e.g., the intron-22 inversion. While some studies have found associations between inhibitor status and promoter polymorphisms in CTLA4, TNFA and/or IL10, others have not. If these promoter polymorphisms are indeed functional and truly influence inhibitor development, their alleles could modulate transcription initiation rates. The goal of this study was to investigate the possibility of cis-acting genotype-specific differences in mean steady-state mRNA levels encoded by CTLA4, TNFA and IL10. Methods: We examined the relationship of lymphocyte CTLA4, TNFA and IL10 mRNA levels with the genotypes of 265 SNPs located across their structural loci in 1189 Mexican American subjects in the San Antonio Family Heart Study. Expression profiles were generated using Illumina's HWG-6 BeadChips and genotypes came from the Illumina OmniExpress-12 BeadChip. Measured genotype association analyses that accounted for non-independence of family members and employed an additive model (in which testing to determine whether gene expression varies by genotype, with the model constrained so that each “dose” of the minor allele raises or lowers gene expression by an equal amount “beta”) were performed using the software package SOLAR. P-values were calculated using a 1 degree-of-freedom chi-square test comparing the likelihood of a model where the change in expression levels by genotype is estimated to the likelihood of a model where beta is constrained to zero. Results: None of the 265 genotyped SNPs within or near these three genes (i.e., 49 SNPs in IL10, 35 SNPs in CTLA4 & 181 SNPs in TNFA) function as cis-acting regulatory variants, as no significant genotype-specific associations with these genes' transcript levels were identified. Conclusions: We observed no evidence for cis-regulation of CTLA4, TNFA or IL10 in Mexican Americans, the largest and most rapidly growing minority population in the United States, despite having genotyped directly the previously implicated promoter polymorphisms in the current analysis (e.g., see Figure). Although Hispanic American HA patients were recently found to have a significantly higher risk for inhibitor development than White HA patients, it is possible that cis-acting functional variants in this minority population are rare and not well-represented by the common GWAS SNPs used for these analyses. Since linkage disequilibrium patterns between markers are population-specific, we are also currently genotyping these SNPs in a large cohort of African and Caucasian American HA patients. Disclosures: No relevant conflicts of interest to declare.

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No Evidence of Cis-Acting SNPs In Immune Response Genes Previously Associated with Factor VIII Inhibitors: The San Antonio Family Heart Study

Blood ◽

10.1182/blood.v116.21.4422.4422 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4422-4422

Author(s):

Tom E. Howard ◽

Kevin R. Viel ◽

Eugene Drigalenko ◽

Shelley Cole ◽

Melinda Epstein ◽

...

Keyword(s):

Immune Response ◽

San Antonio ◽

Expression Profiles ◽

Mrna Levels ◽

Promoter Polymorphisms ◽

Cis Acting ◽

Immune Response Genes ◽

Functional Variants ◽

Family Heart Study ◽

Heart Study

Abstract Abstract 4422 Background/Aims: FVIII gene (F8) mutation type is a risk factor for FVIII inhibitors, which develop in ~20-30% of hemophilia A patients following FVIII replacement therapy. Studies have investigated immune response genes to determine why FVIII is immunogenic in some, but not all, patients with the same F8 abnormality. While some studies have found associations between inhibitor status and promoter polymorphisms in CTLA4, TNFA, and/or IL10, others have not. If these promoter polymorphisms influence inhibitor development, their alleles could modulate transcription initiation rates. The goal of this study was to investigate cis-acting genotype-specific differences in mean steady state mRNA levels. Method: We examined the relationship of lymphocyte CTLA4, TNFA, and IL10 mRNA levels with the genotypes of 49 SNPs located across their structural loci in 1189 Mexican American participants of the San Antonio Family Heart Study (SAFHS). Expression profiles were generated using Illumina's HWG-6 BeadChips and genotypes came from the Illumina Human HapMap 550 SNP panel. Measured genotype association analyses that accounted for non-independence of family members and employed an additive model were performed using the software package SOLAR. Result: Except for C(-13780)A, located upstream of CTLA4, which was modestly associated with CTLA4 mRNA levels (p<0.005), we observed no cis-acting regulatory variants associated with these genes’ transcription levels. Conclusion: We observed little evidence for cis-regulation of CTLA4, TNFA, and IL10 in Mexican Americans. It is possible that cis-acting functional variants are rare and not well-represented by the common GWAS SNPs used for these analyses. Because previously implicated polymorphisms were not included in this analysis and linkage disequilibrium patterns between markers are population-specific, we are currently genotyping these SNPs in the SAFHS. Disclosures: No relevant conflicts of interest to declare.

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Inflammatory and immune response genes: A genetic analysis of inhibitor development in Iranian hemophilia A patients

Pediatric Hematology and Oncology ◽

10.1080/08880018.2019.1585503 ◽

2019 ◽

Vol 36 (1) ◽

pp. 28-39 ◽

Cited By ~ 2

Author(s):

Niloofar Naderi ◽

Hossein Yousefi ◽

Sahar Mollazadeh ◽

Afsaneh Seyed Mikaeili ◽

Masoumeh Keshavarz Norouzpour ◽

...

Keyword(s):

Immune Response ◽

Genetic Analysis ◽

Hemophilia A ◽

Inhibitor Development ◽

Immune Response Genes

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Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2914-2924.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2914-2924

Author(s):

A Hoekema ◽

R A Kastelein ◽

M Vasser ◽

H A de Boer

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Steady State ◽

Codon Usage ◽

Experimental Approach ◽

Mrna Translation ◽

Yeast Cells ◽

Expression Level ◽

Start Codon ◽

Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

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Genotype-dependent and non-gradient patterns of RSV gene expression

10.1101/641878 ◽

2019 ◽

Author(s):

Felipe-Andrés Piedra ◽

Xueting Qiu ◽

Michael N. Teng ◽

Vasanthi Avadhanula ◽

Annette A. Machado ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Steady State ◽

Transcription Initiation ◽

Mrna Levels ◽

Viral Pathogen ◽

Negative Strand ◽

Gene Start ◽

Syncytial Virus ◽

Conserved Gene

AbstractRespiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3’ promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.Author SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3’ promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.

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Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.5016-5025.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 5016-5025

Author(s):

A F Wahl ◽

A M Geis ◽

B H Spain ◽

S W Wong ◽

D Korn ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Steady State ◽

Dna Polymerase ◽

Transformed Cells ◽

Mrna Levels ◽

Dna Polymerase Alpha ◽

Human Dna ◽

Alpha Gene

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

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Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5417 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5417-5425 ◽

Cited By ~ 46

Author(s):

D A Glesne ◽

F R Collart ◽

E Huberman

Keyword(s):

Gene Expression ◽

Steady State ◽

State Level ◽

Mrna Levels ◽

Dependent Manner ◽

Intracellular Level ◽

Nucleotide Analogs ◽

Nucleotide Biosynthesis ◽

Transcriptional Initiation ◽

Imp Dehydrogenase

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

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Regulation of Osteoprotegerin Gene Expression in Dental Follicle Cells

Journal of Dental Research ◽

10.1177/154405910308200411 ◽

2003 ◽

Vol 82 (4) ◽

pp. 298-302 ◽

Cited By ~ 14

Author(s):

G.E. Wise ◽

Y. Ren ◽

S. Yao

Keyword(s):

Gene Expression ◽

Steady State ◽

Protein Kinase ◽

Follicle Cells ◽

Protein Kinase Activity ◽

Mrna Levels ◽

Dental Follicle ◽

Dental Follicle Cells ◽

Parathyroid Hormone Related Protein ◽

Mandibular Molar

Colony-stimulating factor-one (CSF-1) and parathyroid-hormone-related protein (PTHrP) down-regulate osteoprotegerin (OPG) gene expression in the dental follicle of the rat first mandibular molar. To examine this regulation at the signal transduction level, we treated cultured dental follicle cells with either phorbolmyristate acetate (PMA) or dibutyryl cyclic AMP (dbcAMP) to activate either protein kinase C (PKC) or protein kinase A (PKA). Our results demonstrate that PMA up-regulates OPG gene expression and down-regulates the expression of CSF-1 and the PTHrP receptor (PTHrP-R). Conversely, dbcAMP down-regulates OPG expression and up-regulates CSF-1 and PTHrP-R expression. Immunostaining shows that PMA also increases the steady-state levels of protein. Thus, treatment with agents that affect protein kinase activity also enhance the steady-state mRNA and protein levels of OPG, as well as decreasing the mRNA levels of CSF-1 and PTHrP-R. The PKC-α isoform may be critical in OPG regulation because PKC-α gene expression is enhanced by PMA and reduced by either CSF-1 or PTHrP.

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Differential regulation of γ-glutamylcysteine synthetase heavy and light subunit gene expression

Biochemical Journal ◽

10.1042/bj3260167 ◽

1997 ◽

Vol 326 (1) ◽

pp. 167-172 ◽

Cited By ~ 82

Author(s):

Jiaxin CAI ◽

Zong-Zhi HUANG ◽

Shelly C. LU

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Steady State ◽

Mrna Level ◽

Rat Hepatocytes ◽

Mrna Levels ◽

Subunit Gene ◽

Subunit Mrna ◽

Glutamylcysteine Synthetase ◽

Steady State Mrna Level

γ-Glutamylcysteine synthetase (GCS) is the rate-limiting enzyme in the biosynthesis of glutathione and is composed of a heavy and a light subunit. Although the heavy subunit is enzymically active alone, the light subunit plays an important regulatory role by making the holoenzyme function more efficiently. In the current study we examined whether conditions which are known to influence gene expression of the heavy subunit also influence that of the light subunit, and the mechanisms involved. Treatment of cultured rat hepatocytes with hormones such as insulin and hydrocortisone, or plating hepatocytes under low cell density increased the steady-state mRNA level of the heavy subunit only. Treatment with diethyl maleate (DEM), buthionine sulphoximine (BSO) and t-butylhydroquinone (TBH) increased the steady state mRNA level and gene transcription rates of both subunits. These treatments share in common their ability to induce oxidative stress and activate nuclear factor κB (NF-κB). Treatment with protease inhibitors 7-amino-1-chloro-3-tosylamido-2-heptanone (TLCK) or L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) had no influence on the basal NF-κB and GCS subunit mRNA levels, but blocked the activation of NF-κB by DEM, BSO and TBH, and the increase in GCS heavy subunit mRNA level by BSO and TBH. On the other hand, the DEM-, BSO- and TBH-induced increase in GCS light-subunit mRNA level was unaffected by TLCK and TPCK. Thus only the heavy subunit is hormonally regulated and growth sensitive, whereas both subunits are regulated by oxidative stress. Signalling through NF-κB is involved only in the oxidative-stress-mediated changes in the heavy subunit gene expression.

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Genetic Factors Associated with Inhibitor Development in Hemophilia A: Initial Results From the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort.

Blood ◽

10.1182/blood.v114.22.217.217 ◽

2009 ◽

Vol 114 (22) ◽

pp. 217-217 ◽

Cited By ~ 2

Author(s):

Jan Astermark ◽

John Schwarz ◽

Sharyne M. Donfield ◽

Donna M. DiMichele ◽

Bruce M. Ewenstein ◽

...

Keyword(s):

Immune Response ◽

Genetic Factors ◽

Hemophilia A ◽

Laboratory Data ◽

Geographic Region ◽

Outcome Data ◽

Nucleotide Polymorphisms ◽

Severe Hemophilia ◽

Inhibitor Development ◽

Total Group

Abstract Abstract 217 Introduction: Both genetic and environmental factors have been implicated as potential risk factors for the development of inhibitory factor VIII (FVIII) antibodies. Previous studies suggest that genetic factors are of major importance. The causative FVIII mutation likely sets the stage for inhibitor risk, with other genetic markers important in determining the final outcome. Data suggest that the process of inhibitor development is complex, involving a variety of immune regulatory genes, several of which have the potential to modify risk. Through a collaboration among three multi-center studies: the Hemophilia Inhibitor Genetics Study (HIGS), the Malmö International Brother Study (MIBS), and the Hemophilia Growth and Development Study (HGDS), a combined cohort was formed to conduct an association study to test the hypothesis that antibody development to FVIII is mediated by immune response genes. Methods: The study includes clinical and laboratory data for 680 people with hemophilia A. Participants from Europe and North America account for 43% and 57% of the population, respectively. Eighty-five percent have severe (<0.01 IU/mL), 10% moderate (0.01 – 0.05% IU/mL), and 4.4% mild (>0.05 – 0.4 IU/mL) hemophilia. The cohort is predominately Caucasian, 81.0%, with 6.2% of African heritage, 8.8% Hispanic, and the remaining 4% of other races and ethnicities. Forty-nine percent have a current, or history of, an inhibitor ≥1 BU. Using the Illumina iSelect platform, 14,626 single nucleotide polymorphisms (SNPs) from 1,081 candidate genes were genotyped. These included immune response and immune modifier genes: cytokines and their receptors, chemokines and their receptors, and pathway genes involved in inflammatory and immune responses. Analyses were completed among the total group and the subgroup with severe hemophilia to identify SNPs associated with inhibitor status. The models were adjusted for population admixture, severity of hemophilia, type of mutation (high vs. low risk), year of birth, and geographic region. Meta analyses were used to obtain single odds ratios (OR) and p-values for the three cohorts. Results: 13,952 of the 14,626 (95.4%) SNPs were successfully genotyped. One hundred fourteen were associated with inhibitor status at the p<0.01 level. Strong SNP associations for the total group were observed in the DOCK2 (OR 0.28, p= 0.00004, and OR 3.9, p=0.0002), MAPK9 (OR 2.0, p=0.0003), F13A1 (OR 0.32, p=0.00007), CD36 (OR 0.56, p=0.0002), and PTPRR (OR 0.51, p=0.0003) genes. For four markers located within the MAPK9, DOCK2, and CD36 genes, the associations were similar, or stronger, for the subgroup with severe hemophilia. Analysis of polymorphisms in the FVIII gene, completion of HLA Class II typing, and haplotype analysis are underway. Conclusions: Our findings suggest that functional pathways involved in a variety of cellular processes will be important in inhibitor development, but these results warrant further study and replication in similarly powered case-control or cohort studies. Disclosures: Ewenstein: Baxter Healthcare: Employment. Spotts:Baxter Healthcare: Employment.

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