Epstein-Barr Virus-transformed Lymphocytes Research Articles

Multi-omics analysis reveals novel causal pathways in psoriasis pathogenesis

BackgroundTo elucidate the genetic and molecular mechanisms underlying psoriasis by employing an integrative multi-omics approach, using summary-data-based Mendelian randomization (SMR) to infer causal relationships among DNA methylation, gene expression, and protein levels in relation to psoriasis risk.MethodsWe conducted SMR analyses integrating genome-wide association study (GWAS) summary statistics with methylation quantitative trait loci (mQTL), expression quantitative trait loci (eQTL), and protein quantitative trait loci (pQTL) data. Publicly available datasets were utilized, including psoriasis GWAS data from the European Molecular Biology Laboratory–European Bioinformatics Institute and the UK Biobank. Heterogeneity in dependent instruments (HEIDI) test and colocalization analyses were performed to identify shared causal variants, and multi-omics integration was employed to construct potential regulatory pathways.ResultsOur analyses identified significant causal associations between DNA methylation, gene expression, protein abundance, and psoriasis risk. We discovered two pathways involving the long non-coding RNA RP11-977G19.11 and apolipoprotein F (APOF). Methylation at sites cg26804944 and cg02705573 was negatively associated with RP11-977G19.11 expression. Reduced expression of RP11-977G19.11 was linked to increased APOF levels, which were positively associated with a higher risk of psoriasis. Methylation at sites cg00172967, cg00294382, and cg24773560 was positively associated with RP11-977G19.11 expression. Elevated expression of RP11-977G19.11 was associated with decreased APOF levels, reducing the risk of psoriasis. Colocalization analysis highlighted APOF as a key protein in psoriasis pathogenesis. Validation using skin tissue, EBV-transformed lymphocytes data and inflammation-related protein panels confirmed the associations of RP11-977G19.11 and APOF with psoriasis.ConclusionsOur multi-omics analysis provides preliminary evidence for potential molecular mechanisms in psoriasis pathogenesis. Through the integration of GWAS and molecular QTL data, we identify candidate pathways that may be relevant to disease biology. While these findings require extensive experimental validation, they offer a framework for future investigations into the molecular basis of psoriasis.

Meta-analyses uncover the genetic architecture of Idiopathic Inflammatory Myopathies.

Idiopathic inflammatory myopathies (myositis, IIMs) are rare, systemic autoimmune disorders that lead to muscle inflammation, weakness, and extra-muscular manifestations, with a strong genetic component influencing disease development and progression. Previous genome-wide association studies identified loci associated with IIMs. In this study, we imputed data from two prior genome-wide myositis studies and analyzed the largest myositis dataset to date to identify novel risk loci and susceptibility genes associated with IIMs and its clinical subtypes. We performed association analyses on 14,903 individuals (3,206 cases and 11,697 controls) with genotypes and imputed data from the Trans-Omics for Precision Medicine (TOPMed) reference panel. Fine-mapping and expression quantitative trait locus co-localization analyses in myositis-relevant tissues indicated potential causal variants. Functional annotation and network analyses using the random walk with restart (RWR) algorithm explored underlying genetic networks and drug repurposing opportunities. Our analyses identified novel risk loci and susceptibility genes, such as FCRLA, NFKB1, IRF4, DCAKD, and ATXN2 in overall IIMs; NEMP2 in polymyositis; ACBC11 in dermatomyositis; and PSD3 in myositis with anti-histidyl-tRNA synthetase autoantibodies (anti-Jo1). We also characterized effects of HLA region variants and the role of C4. Colocalization analyses suggested putative causal variants in DCAKD in skin and muscle, HCP5 in lung, and IRF4 in EBV-transformed lymphocytes, lung, and whole blood. RWR further prioritized additional candidate genes, including APP, CD74, CIITA, NR1H4, and TXNIP, for future investigation. Our study uncovers novel genetic regions contributing to IIMs, advancing our understanding of myositis pathogenesis and offering new insights for future research.

Susceptibility gene identification and risk evaluation model construction by transcriptome-wide association analysis for salt sensitivity of blood pressure

BackgroundSalt sensitivity of blood pressure (SSBP) is an intermediate phenotype of hypertension and is a predictor of long-term cardiovascular events and death. However, the genetic structures of SSBP are uncertain, and it is difficult to precisely diagnose SSBP in population. So, we aimed to identify genes related to susceptibility to the SSBP, construct a risk evaluation model, and explore the potential functions of these genes.Methods and resultsA genome-wide association study of the systemic epidemiology of salt sensitivity (EpiSS) cohort was performed to obtain summary statistics for SSBP. Then, we conducted a transcriptome-wide association study (TWAS) of 12 tissues using FUSION software to predict the genes associated with SSBP and verified the genes with an mRNA microarray. The potential roles of the genes were explored. Risk evaluation models of SSBP were constructed based on the serial P value thresholds of polygenetic risk scores (PRSs), polygenic transcriptome risk scores (PTRSs) and their combinations of the identified genes and genetic variants from the TWAS. The TWAS revealed that 2605 genes were significantly associated with SSBP. Among these genes, 69 were differentially expressed according to the microarray analysis. The functional analysis showed that the genes identified in the TWAS were enriched in metabolic process pathways. The PRSs were correlated with PTRSs in the heart atrial appendage, adrenal gland, EBV-transformed lymphocytes, pituitary, artery coronary, artery tibial and whole blood. Multiple logistic regression models revealed that a PRS of P < 0.05 had the best predictive ability compared with other PRSs and PTRSs. The combinations of PRSs and PTRSs did not significantly increase the prediction accuracy of SSBP in the training and validation datasets.ConclusionsSeveral known and novel susceptibility genes for SSBP were identified via multitissue TWAS analysis. The risk evaluation model constructed with the PRS of susceptibility genes showed better diagnostic performance than the transcript levels, which could be applied to screen for SSBP high-risk individuals.

Abstract PO5-09-03: Multi-tissue transcriptome-wide association studies identified genes for intrinsic subtypes of breast cancer

Abstract Background: Though several genome-wide association studies (GWAS) of breast cancer (BC) have identified common variants which differ between intrinsic subtypes, the genes through which these variants act through to impact BC risk have not been fully established. Furthermore, transcriptome-wide association studies (TWAS) have thus far been primarily employed to identify genes associated with overall BC risk, while overlooking how the influence of common variation on gene expression may contribute to subtype-specific differences. Methods: In this study, we performed two complementary multi-tissue TWASs for each of the following BC intrinsic subtypes: Luminal A-like, Luminal B-like, Luminal B/HER2-negative-like, HER2-enriched-like, Triple Negative BC. These two approaches included 1) an expression-based approach that collated TWAS signals from expression quantitative trait loci (eQTLs) across multiple tissues using the Aggregated Cauchy Association Test (ACAT) and 2) a splicing-based approach that utilized two applications of ACAT to collate splicing QTLs (sQTLs) for a given gene in a tissue and then across tissues. To perform these two TWASs, we utilized e/sQTL models trained in 11 tissues from the Genotype-Tissue Expression Project including breast, ovary, uterus, vagina, EBV-transformed lymphocytes, whole blood, spleen, liver, subcutaneous adipose, visceral adipose, and cell-cultured fibroblasts. GWAS summary statistics were previously generated from 133,384 BC cases and 113,789 controls who were participants in the Breast Cancer Association Consortium (BCAC). We further performed our TWAS while conditioning e/sQTL effect sizes on nearby GWAS index SNPs. Additionally, we utilized gene-based fine-mapping of eQTLs and sQTL to identify candidate causal genes for each intrinsic subtype. Results: Overall, we identified 164 genes in 69 loci that were associated with Luminal A-like, 19 genes in 9 loci with Luminal B-like, 18 genes in 11 loci with Luminal B/HER2-negative-like, 10 genes in 7 loci with HER2-enriched-like, and 29 genes in 12 loci with TNBC. Among these genes, 17 genes had not been reported in previous TWAS of BC, and 140 genes, 1 gene, 2 genes, 2 genes, and 16 genes were uniquely associated with each of the intrinsic subtypes, respectively. Additionally, we identified one gene associated with Luminal A-like and one gene with Luminal B-like BC that were each in a locus located at least 1.4 Mb from published GWAS hits. Furthermore, we identified 106, 11, 10, 5, and 21 candidate causal genes for each of the intrinsic subtypes, respectively, that had a posterior inclusion probability of 0.9 in at least one e/sQTL. Conclusion: In summary, our multi-tissue TWAS corroborated previous GWAS loci for overall BC risk and intrinsic subtypes, while underscoring how common variation impacts BC etiology by modulating the expression and splicing of genes in multiple tissue types. Citation Format: James Li, Julian McClellan, Haoyu Zhang, Guimin Gao, Dezheng Huo. Multi-tissue transcriptome-wide association studies identified genes for intrinsic subtypes of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-09-03.

Shared genetic architecture between autoimmune disorders and B-cell acute lymphoblastic leukemia: insights from large-scale genome-wide cross-trait analysis.

To study the shared genetic structure between autoimmune diseases and B-cell acute lymphoblastic leukemia (B-ALL) and identify the shared risk loci and genes and genetic mechanisms involved. Based on large-scale genome-wide association study (GWAS) summary-level data sets, we observed genetic overlaps between autoimmune diseases and B-ALL, and cross-trait pleiotropic analysis was performed to detect shared pleiotropic loci and genes. A series of functional annotation and tissue-specific analysis were performed to determine the influence of pleiotropic genes. The heritability enrichment analysis was used to detect crucial immune cells and tissues. Finally, bidirectional Mendelian randomization (MR) methods were utilized to investigate the casual associations. Our research highlighted shared genetic mechanisms between seven autoimmune disorders and B-ALL. A total of 73 pleiotropic loci were identified at the genome-wide significance level (P < 5 × 10-8), 16 of which had strong evidence of colocalization. We demonstrated that several loci have been previously reported (e.g., 17q21) and discovered some novel loci (e.g., 10p12, 5p13). Further gene-level identified 194 unique pleiotropic genes, for example IKZF1, GATA3, IKZF3, GSDMB, and ORMDL3. Pathway analysis determined the key role of cellular response to cytokine stimulus, B cell activation, and JAK-STAT signaling pathways. SNP-level and gene-level tissue enrichment suggested that crucial role pleiotropic mechanisms involved in the spleen, whole blood, and EBV-transformed lymphocytes. Also, hyprcoloc and stratified LD score regression analyses revealed that B cells at different developmental stages may be involved in mechanisms shared between two different diseases. Finally, two-sample MR analysis determined causal effects of asthma and rheumatoid arthritis on B-ALL. Our research proved shared genetic architecture between autoimmune disorders and B-ALL and shed light on the potential mechanism that might involve in.

Evidence for the gut-skin axis: Common genetic structures in inflammatory bowel disease and psoriasis.

Inflammatory bowel disease (IBD) and psoriasis (Ps) are common immune-mediated diseases that exhibit clinical comorbidity, possibly due to a common genetic structure. However, the exact mechanism remains unknown. The study population consisted of IBD and Ps genome-wide association study (GWAS) data. Genetic correlations were first evaluated. Then, the overall evaluation employed LD score regression (LDSC), while the local assessment utilized heritability estimation from summary statistics (HESS). Causality assessment was conducted through two-sample Mendelian randomization (2SMR), and genetic overlap analysis utilized the conditional false discovery rate/conjunctional FDR (cond/conjFDR) method. Finally, LDSC applied to specifically expressed genes (LDSC-SEG) was performed at the tissue level. For IBD and Ps-specific expressed genes, genetic correlation, causality, shared genetics, and trait-specific associated tissues were methodically examined. At the genomic level, both overall and local genetic correlations were found between IBD and Ps. MR analysis indicated a positive causal relationship between Ps and IBD. The conjFDR analysis with a threshold of<0.01 identified 43 loci shared between IBD and Ps. Subsequent investigations into disease-associated tissues indicated a close association of IBD and Ps with whole blood, lung, spleen, and EBV-transformed lymphocytes. The current research offers a novel perspective on the association between IBD and Ps. It contributes to an enhanced comprehension of the genetic structure and mechanisms of comorbidities in both diseases.

Abstract 5225: Expression- and splicing-based multi-tissue transcriptome-wide association studies identified multiple genes for estrogen receptor-negative breast cancer

Abstract Although several transcriptome-wide association studies (TWAS) have been performed to identify genes associated with overall breast cancer risk, few genes were found to be associated with estrogen receptor-negative (ER-) breast cancer. These studies were based on gene expression prediction models trained mainly in breast tissue, and they did not account for alternative splicing of genes. In this study, we utilized two approaches to perform multi-tissue TWASs of ER- breast cancer: 1) an expression-based TWAS that combined TWAS signals for each gene across multiple tissues and 2) a splicing-based TWAS that combined TWAS signals of all excised introns for each gene across tissues. To perform these two TWASs, we first obtained gene expression prediction models that were trained in 11 tissues from the Genotype-Tissue Expression including breast, ovary, uterus, vagina, EBV-transformed lymphocytes, whole blood, spleen, liver, subcutaneous adipose, visceral adipose, and cell-cultured fibroblasts. Furthermore, we obtained GWAS summary statistics for ER- breast cancer by performing a meta-analysis of 21,468 ER- cases and 105,974 controls from the Breast Cancer Association Consortium and 9414 BRCA1 mutation carriers who were breast cancer cases and 9494 BRCA1 mutation carriers who did not have breast cancer from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. For our expression-based TWAS, we utilized the Aggregated Cauchy Association Test (ACAT) to collate TWAS signals across all 11 tissues for each gene using equal weights for each tissue, while for our splicing-based TWAS, we utilized ACAT to collate TWAS signals for all excised introns in all tissues for each gene. Overall, we identified 63 genes in 29 loci that were significantly associated with ER- breast cancer, including 23 genes that were identified using both approaches, 27 that were uniquely identified in the expression-based TWAS, and 13 that were uniquely identified in the splicing-based TWAS. Of the 63 genes, 50 genes have not been previously reported in TWAS studies of breast cancer. Using a weighted ACAT method, in which breast tissue was given a 5-fold higher weight, we obtained similar results. In summary, our joint, multi-tissue TWAS corroborated previous GWAS loci for both ER- and overall breast cancer while highlighting how incorporating TWAS signals from multiple tissues and alternative splicing allowed us to discover new susceptibility genes for ER- breast cancer. Citation Format: James L. Li, Julian C. McClellan, Guimin Gao, Dezheng Huo. Expression- and splicing-based multi-tissue transcriptome-wide association studies identified multiple genes for estrogen receptor-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5225.

Integrative Analysis of Transcriptome-Wide Association Study and Gene-Based Association Analysis Identifies In Silico Candidate Genes Associated with Juvenile Idiopathic Arthritis.

Genome-wide association study (GWAS) of Juvenile idiopathic arthritis (JIA) suffers from low power due to limited sample size and the interpretation challenge due to most signals located in non-coding regions. Gene-level analysis could alleviate these issues. Using GWAS summary statistics, we performed two typical gene-level analysis of JIA, transcriptome-wide association studies (TWAS) using FUnctional Summary-based ImputatiON (FUSION) and gene-based analysis using eQTL Multi-marker Analysis of GenoMic Annotation (eMAGMA), followed by comprehensive enrichment analysis. Among 33 overlapped significant genes from these two methods, 11 were previously reported, including TYK2 (PFUSION = 5.12 × 10-6, PeMAGMA = 1.94 × 10-7 for whole blood), IL-6R (PFUSION = 8.63 × 10-7, PeMAGMA = 2.74 × 10-6 for cells EBV-transformed lymphocytes), and Fas (PFUSION = 5.21 × 10-5, PeMAGMA = 1.08 × 10-6 for muscle skeletal). Some newly plausible JIA-associated genes are also reported, including IL-27 (PFUSION = 2.10 × 10-7, PeMAGMA = 3.93 × 10-8 for Liver), LAT (PFUSION = 1.53 × 10-4, PeMAGMA = 4.62 × 10-7 for Artery Aorta), and MAGI3 (PFUSION = 1.30 × 10-5, PeMAGMA = 1.73 × 10-7 for Muscle Skeletal). Enrichment analysis further highlighted 4 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 10 Gene Ontology (GO) terms. Our findings can benefit the understanding of genetic determinants and potential therapeutic targets for JIA.

Transcriptome-wide association study identifies susceptibility genes for rheumatoid arthritis

ObjectiveTo identify rheumatoid arthritis (RA)-associated susceptibility genes and pathways through integrating genome-wide association study (GWAS) and gene expression profile data.MethodsA transcriptome-wide association study (TWAS) was conducted by the FUSION software for RA considering EBV-transformed lymphocytes (EL), transformed fibroblasts (TF), peripheral blood (NBL), and whole blood (YBL). GWAS summary data was driven from a large-scale GWAS, involving 5539 autoantibody-positive RA patients and 20,169 controls. The TWAS-identified genes were further validated using the mRNA expression profiles and made a functional exploration.ResultsTWAS identified 692 genes with PTWAS values < 0.05 for RA. CRIPAK (PEL = 0.01293, PTF = 0.00038, PNBL = 0.02839, PYBL = 0.0978), MUT (PEL = 0.00377, PTF = 0.00076, PNBL = 0.00778, PYBL = 0.00096), FOXRED1 (PEL = 0.03834, PTF = 0.01120, PNBL = 0.01280, PYBL = 0.00583), and EBPL (PEL = 0.00806, PTF = 0.03761, PNBL = 0.03540, PYBL = 0.04254) were collectively expressed in all the four tissues/cells. Eighteen genes, including ANXA5, AP4B1, ATIC (PTWAS = 0.0113, downregulated expression), C12orf65, CMAH, PDHB, RUNX3 (PTWAS = 0.0346, downregulated expression), SBF1, SH2B3, STK38, TMEM43, XPNPEP1, KIAA1530, NUFIP2, PPP2R3C, RAB24, STX6, and TLR5 (PTWAS = 0.04665, upregulated expression), were validated with integrative analysis of TWAS and mRNA expression profiles. TWAS-identified genes functionally involved in endoplasmic reticulum organization, regulation of cytokine production, TNF signaling pathway, immune response-regulating signaling pathway, regulation of autophagy, etc.ConclusionWe identified multiple candidate genes and pathways, providing novel clues for the genetic mechanism of RA.

Lifelong Reduction in LDL (Low-Density Lipoprotein) Cholesterol due to a Gain-of-Function Mutation in LDLR.

Loss-of-function mutations in the LDL (low-density lipoprotein) receptor gene (LDLR) cause elevated levels of LDL cholesterol and premature cardiovascular disease. To date, a gain-of-function mutation in LDLR with a large effect on LDL cholesterol levels has not been described. Here, we searched for sequence variants in LDLR that have a large effect on LDL cholesterol levels. We analyzed whole-genome sequencing data from 43 202 Icelanders. Single-nucleotide polymorphisms and structural variants including deletions, insertions, and duplications were genotyped using whole-genome sequencing-based data. LDL cholesterol associations were carried out in a sample of >100 000 Icelanders with genetic information (imputed or whole-genome sequencing). Molecular analyses were performed using RNA sequencing and protein expression assays in Epstein-Barr virus-transformed lymphocytes. We discovered a 2.5-kb deletion (del2.5) overlapping the 3' untranslated region of LDLR in 7 heterozygous carriers from a single family. Mean level of LDL cholesterol was 74% lower in del2.5 carriers than in 101 851 noncarriers, a difference of 2.48 mmol/L (96 mg/dL; P=8.4×10-8). Del2.5 results in production of an alternative mRNA isoform with a truncated 3' untranslated region. The truncation leads to a loss of target sites for microRNAs known to repress translation of LDLR. In Epstein-Barr virus-transformed lymphocytes derived from del2.5 carriers, expression of alternative mRNA isoform was 1.84-fold higher than the wild-type isoform (P=0.0013), and there was 1.79-fold higher surface expression of the LDL receptor than in noncarriers (P=0.0086). We did not find a highly penetrant detrimental impact of lifelong very low levels of LDL cholesterol due to del2.5 on health of the carriers. Del2.5 is the first reported gain-of-function mutation in LDLR causing a large reduction in LDL cholesterol. These data point to a role for alternative polyadenylation of LDLR mRNA as a potent regulator of LDL receptor expression in humans.

Bioinformatic analysis of differentially expressed genes and identification of key genes in EBV-transformed lymphoblasts

Although the Epstein-Barr virus (EBV) is a well-known human oncogenic virus, its molecular mechanisms involved in the transformation of healthy human cells remain poorly understood. In this study, human lymphocytes were isolated from the peripheral blood of healthy adults, and lymphocytes were transformed in vitro by EBV. Agilent human whole genome microarrays were used to detect the differential gene expression profiles of EBV-transformed lymphoblasts and healthy peripheral blood lymphocytes (PBLs). By constructing the gene functional network of EBV-induced lymphocyte transformation, we screened out candidate key genes in this process and verified their expression levels by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In the EBV-transformed lymphoblasts, 2335 differentially expressed genes, including 1328 up-regulated and 1007 down-regulated, were screened out. Five candidate key genes, namely, PLK1, E2F1, PTPN11, BIRC5 and FYN were mainly screened out according to the results of LIMMA, String, Cytoscape software analysis. RT-qPCR and Western blot showed that PLK1, E2F1, PTPN11, BIRC5 genes had increased expression levels, and FYN gene was down-regulated in EBV-transformed lymphoblasts. Silencing of PLK1 gene in Raji cells could inhibit cell proliferation and invasion, and induce cell cycle arrest and apoptosis. In conclusion, PLK1, E2F1, PTPN11, BIRC5 and FYN are the candidate key molecules of EBV-transformed lymphocytes.

Genetic associations and expression of extra-short isoforms of disrupted-in-schizophrenia 1 in a neurocognitive subgroup of schizophrenia.

Disrupted-in-schizophrenia 1 (DISC1) was reported to be associated with schizophrenia. In a previous study, we found significant association with schizophrenia patients with deficient sustained attention assessed by continuous performance test (CPT). This study aimed to identify risk polymorphisms in this specific neurocognitive subgroup and investigate the expression of different isoforms of DISC1. A total of 83 genetic variants were identified through direct sequencing in 50 controls and 100 schizophrenia patients. Fourteen variants were genotyped in 600 controls and 912 patients. Patients were subgrouped by familial loading (multiplex or simplex) and performance on CPT. The frequency of AA genotype of rs11122324 at the 3'-UTR of Es and Esv1 isoforms and of rs2793091 at intron 4 were significantly higher in multiplex schizophrenia patients than those in controls (corrected p < 0.05). In further subgrouping, the frequency of AA genotype of the two SNPs were significantly higher in multiplex schizophrenia patients with deficient sustained attention than those in controls (corrected p < 0.005). The mRNA expression levels of two extra-short isoforms (Es and Esv1) in the EBV-transformed lymphocytes of schizophrenia were significantly higher than those of controls. Luciferase reporter assays demonstrated that the A-allele of rs11122324 significantly upregulated DISC1 extra-short isoforms transcription compared with the G-allele. We found two SNPs (rs11122324 and rs2793091) of DISC1 may be specifically associated with multiplex schizophrenia patients with deficient sustained attention. The SNP rs11122324 may be a risk polymorphism, which may have functional influence on the transcription of Es and Esv1 through increasing their expression.

Association of MCP-1 promoter polymorphism with susceptibility to nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is prevalent among populations from southern China and is influenced by both genetic and environmental risk factors. The monocyte chemoattractant protein-1 (MCP-1), a member of cysteine-cysteine chemokine family, plays critical roles in cancers. A polymorphism within the MCP-1 promoter, rs1024611, has been shown to be significantly associated with the risk of several cancers. Our purpose was to assess the role of rs1024611 in NPC susceptibility. By polymerase chain reaction-restriction fragment length polymorphism method, we genotyped rs1024611 in 593 patients with NPC (cases) and 480 cancer-free subjects (controls) among Guangxi population from southern China. We observed that the G allele of rs1024611 was significantly associated with the increased risk of NPC in an additive model and dominant model, respectively (P = 0.018 and 0.010, odds ratio = 1.25 and 1.41, respectively). No appreciable variation of the effects was found across the subgroups stratified by age, sex, nationality, smoking and drinking status, and smoking level. In addition, significantly higher messenger RNA (mRNA) expression level of MCP-1 was observed in NPC tissues than that in normal nasopharyngeal tissues, and the G allele of rs1024611 was significantly associated with elevated mRNA expression level of MCP-1 in Epstein-Barr virus-transformed lymphocytes. In conclusion, our findings suggested that rs1024611 at the MCP-1 promoter may be a risk factor for NPC. Further studies with larger sample size are necessary to confirm these findings.

Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits.

D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia.

Effect of Interleukins on Antibody Production by Epstein-Barr Virus Transformed B Cells.

During the past few decades, monoclonal antibodies (MAbs) have become an increasingly used tool in diagnostics, therapeutics, and biomedical research. Several methods have been employed to produce MAbs, one of which is the immortalization of B cells by Epstein-Barr virus (EBV). Despite its simplicity, this procedure was never routinely adopted due to its poor efficiency and short-lived antibody (Ab) production. Various adjustments to the basic procedure were introduced, including the addition of certain cytokines and CpG oligodeoxynucleotides, which were shown to improve EBV infectivity and cloning efficiency. The objective of this study was to manipulate culture conditions of the EBV-transformed human lymphocytes, lymphoblastoid cell lines (LCLs), by the timely addition of stimuli including CpG and various interleukins. Such manipulations are aimed at improving LCL proliferative activity and enhancing the cell lines' immortalization potential as well as their Ab production. To accomplish this, IgG(+) B cells were isolated from peripheral blood of a hepatitis B vaccinated, anti-HB Ab-positive volunteer. These cells were infected with EBV and incubated in the presence of CpG DNA 2006 motifs, recombinant human interleukin-2 (rhIL-2), rhIL-4, rhIL-6, and rhIL-21, individually and in combinations. Cells were then restimulated for 2 weeks with the same ILs. The effect of these ILs on anti-HB Ab production and the proliferation of the EBV-transformed lymphocytes were investigated. The current study demonstrates that treatment of LCL cultures with rhIL-2, rh-IL4, rhIL-6, and rhIL-21, individually and in combination, increased to varying degrees the proliferative activity and Ab production of these cells. The addition of IL-4 alone was able to sustain increase in anti-HB Ab despite IL-4 withdrawal. This study suggests that with further optimization ILs can have an enhancing effect on LCL immortalization potential and Ab production capacity.

Expression of latent membrane proteins in Epstein‑Barr virus-transformed lymphocytes invitro.

Infection with Epstein-Barr virus (EBV) induces activation and proliferation of Blymphocytes. Detection of latent membrane protein (LMP)-1 is used to identify the proliferative ability of B cells. However, changes in the expression levels of the three LMPs during EBV-induced Blymphocyte transformation, have not yet been reported. In the present study, the expression levels of LMP-1, LMP-2A and LMP-2B were compared between EBV-transformed Blymphocytes and paired normal lymphocytes. Seven lymphoblast cell lines were established by EBV infection of normal human lymphocytes invitro. The expression levels of LMP genes and LMP-1 protein were determined using quantitative (q)PCR and western blotting in lymphoblasts and normal lymphocytes, respectively. The expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts compared with the normal lymphocytes. The LMP-1 protein level was also significantly increased in EBV-transformed B lymphocytes. Expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts, suggesting LMP genes are important in the transformation of human lymphocytes.

HOGG1-Cys326 variant cells are hypersensitive to DNA repair inhibition by nitric oxide

The repair of 8-oxo-7,8-dihydroguanine in the DNA of mammalian cells is initiated by 8-oxoguanine DNA glycosylase (OGG1). A frequent polymorphism in the human OGG1 gene, rs1052133, causes the substitution of serine by cysteine at amino acid 326 of the protein and has been associated with an altered risk for various types of cancer in some populations. The OGG1-Cys326 protein appears to have normal enzymatic activity, but greater sensitivity to oxidation than the serine variant. Here, we describe a comparison of the cellular repair by the two OGG1 variants under stress conditions characteristic of inflammation, namely in cells pretreated with nitric oxide (NO) or pre-exposed to hyperthermia. The results show that NO at concentrations causing negligible DNA damage and little cytotoxicity strongly reduces the repair rates of oxidized purines in the DNA of HeLa cells overexpressing the OGG1-Cys326 variant. The reduction in repair was much less pronounced in isogenic cells overexpressing the OGG1-Ser326 variant. Similar results were observed in EBV-transformed lymphocytes from donors homozygous for the two OGG1 variant alleles. In contrast, hyperthermia-induced stress caused a repair retardation that was independent of the OGG1 polymorphism. The repair inhibition by NO in the variant cells gave rise to increased genetic instability, measured as increased micronuclei formation after oxidant exposure. The results could explain a higher risk of malignant transformation in inflamed tissues of carriers of this variant allele.

Genetic Characterization Of Williams Beuren Syndrome Associated With Non-Hodgkin Lymphoma

IntroductionConstitutional chromosomal abnormalities are known to play a role in oncogenesis as shown by the increased incidence of leukemias in children with Down syndrome. Williams Beuren syndrome (WBS, OMIM#194050) is a rare multisystem disorder associating cardiovascular malformations, psychomotor and behavioral abnormalities, a characteristic facial dysmorphism and endocrine defects. WBS is caused by a hemizygous microdeletion on chromosome 7q11.23 spanning 1.5 Mb and encompassing 28 genes and 2 miRNAs. The incidence of typical forms is 1/20, 000 births. Malignancies are not included in the phenotype of this syndrome. However, a thorough review of the literature shows that 12 cases of cancer have been reported in these patients and strikingly 6 of them were Non Hodgkin Lymphoma (NHL). We studied 3 novel cases of cancer including 2 NHL and one esophageal adenocarcinoma (EAC) occurring in WBS patients, in order to characterize genetic abnormalities explaining the susceptibility to cancer in this genetic disease. MethodsWe characterized the microdeletion size and other chromosomal aberrations in the 3 WBS patients using Comparative Genomic Hybridization array. Next Generation Sequencing of the 28 genes and the 2 miRNA loci was performed. The mRNA and miRNA relative expression of tumor tissue and healthy material was investigated by quantitative real time PCR. Finally, DNA repair deficiency was also assessed by microsatellite instability analysis and UV-induced gammaH2AX production in EBV-transformed lymphocytes derived from PBMCs of 2 WBS patients. ResultsThe CGH-array analysis did not identify a recurrent chromosomal rearrangement and there was no evidence of recurrent mutation or biallelic deletion in the WBS genes or miRNA coding sequences. The WBS deletion had a typical 1.5 Mb length for the 2 patients diagnosed with NHL but the patient who presented an EAC had a larger deletion (15 Mb). Lymphoma cells of the first WBS patient showed a common biallelic deletion ofCDKN2A and the second WBS patient showed an atypical deletion of IKZF2. As far as we know, this deletion had never been described in NHL. The esophageal adenocarcinoma cells of the third WBS patient presented 2 amplifications flanking the WBS deletion and involving EGFR and CDK6. These amplifications had the same intensity and we may suggest that they resulted from a double minute formation. The microsatellites were found to be stable and gammaH2AX formation was quite similar in B-EBV transformed lymphocytes derived from WBS patients and healthy controls. As expected, mRNA expression of most of the WBS genes was diminished by approximately a half in normal tissues.Interestingly the expression of WSTF, GTF2IRD2, CLIP2 and STX1Aand the expression of hsa-mir-590 were found to be moreprofoundlydecreased in the tumor cells of the 3 WBS patients. DiscussionA number of genes deleted in WBS (PMS2L, WSTF, RFC2 and TFII-I family genes) are implicated in DNA repair pathways and may represent tumor suppressor genes. Furthermore, the atypical distribution of cancers in WBS is suggestive of a genomic instability associated cancer syndrome. Savina et al (Mutation research, 2011) confirmed experimentally the relationship between an abnormal DNA-damage response and the 7q11.23 hemizygous microdeletion when comparing the comet assay data in FISH-positive and FISH-negative lymphocytes from WBS-suspected patients. However, in our study, no genomic instability was found. Predisposition to NHL is rare and most often associated with inherited immunodeficiency and bone marrow failure syndrome (e.g. Wiskott-Aldrich syndrome, Bloom syndrome, Nijmegen Breakage syndrome). In the present study we focused on a possible link between WBS and NHL. The risk of cancer in this population needs to be accurately evaluated by an international epidemiological study, but the high proportion of NHL is of considerable interest given the scarcity of NHL in young children and the lack of current knowledge on NHL predisposing genes in children without immunodeficiency. NHL was associated with significant underexpression of WSTF, GTF2IRD2, CLIP2, STX1A and has-mir-590. Further investigations are needed to elucidate the molecular pathways involved in NHL initiation in this population. In addition, we propose the constitution of a clinical and biological database of WBS associated cancer cases. Disclosures:No relevant conflicts of interest to declare.

CBX2 gene analysis in patients with 46,XY and 46,XX gonadal disorders of sex development

To investigate a cohort of patients with gonadal disorders of sex development (DSD) for causative CBX2 gene mutations and or gene copy number changes. Genetic association study. University laboratory and tertiary university-based referral center. 47 patients with different forms of 46,XY or 46,XX gonadal DSD. CBX2 gene sequencing and development of a synthetic probe set for multiplex ligation probe amplification (MLPA) to detect CBX2 copy number changes, and reverse-transcriptase polymerase chain reaction (RT-PCR) to evaluate CBX2 expression in two different cell-line types. Gene sequence alteration and or partial or complete gene copy number variations, and detection of CBX2 mRNA isoforms. We detected 10 sequence alterations, 9 reported single nucleotide polymorphisms (SNPs), and a previously unreported variant. This was a silent c.1356G>A transition that may represent a normal variant. A rare SNP (c.1411C>G, p.471Pro>Ala) was found in heterozygous form in one patient. No deletions or duplications were detected by MLPA. Expression of both CBX2 mRNA isoforms was documented in gonadal fibroblasts and Epstein Barr virus (EBV)-transformed lymphocytes. No pathogenic CBX2 mutation was detected. Both CBX2 isoforms are expressed in gonadal fibroblasts and EBV-transformed lymphocytes. This study does not support CBX2 gene disruption as a common cause of gonadal DSD.

Young‐onset parkinsonism due to homozygous duplication of α‐synuclein in a consanguineous family

With a growing list of monogenetic causes of Parkinson's disease (PD),1–3 prioritization of genetic testing is made on the presumed mode of inheritance. In patients with early-onset PD from consanguineous families, the most common mode of inheritance is autosomal recessive. Rarely, an individual may be homozygous for a mutation inherited in an autosomal dominant manner, complicating the diagnostic process. The proband is a 36-year-old female from a consanguineous Pakistani family (Fig. 1A). She presented with a 5-year history of left-sided stiffness, micrographia, festinant gait, tremor, and falls. Past medical history included postpartum psychosis and severe depression. There were no symptoms of autonomic dysfunction. Neurological examination at the age of 35 years revealed severe bilateral bradykinesia, rigidity, worse on the left, and mild bilateral upper limb rest tremor. Gait was shuffling, with freezing and complete loss of postural reflexes. Bilateral ankle clonus was noted. Initially, there was a poor response to dopamine agonists. She responded well to 100/25 mg co-careldopa 3 times per day, but developed wearing offs and peak-dose dyskinesias after 4 months of treatment. There was no family history of parkinsonism. The proband's mother's neurological examination at the age of 72 years was normal. The proband's father, who died at age 64 years from a stroke, had no symptoms or signs of parkinsonism as described by his family. Her siblings and children were asymptomatic, but were unavailable for examination. Figure 1 A: Pedigree and clinical information on the family. A half filled symbol represents the proband (arrow), who is the only individual affected by parkinsonism. Clinically unaffected but confirmed mutation carrier (the proband's mother) is represented by ... Investigations in the proband included routine blood work, copper studies, and white cell enzymes, and were all normal. Dopamine transporter scan (DAT-SCAN) showed bilateral reduction in tracer uptake in the striatum, more significant on the right. Formal neuropsychological examination revealed a mild degree of intellectual under-functioning from premorbid estimates, with anterior/subcortical dysfunction. Mutations in PARKIN, PINK1, and common LRRK2 mutations were excluded. Multiplex ligation-dependent probe amplification (MLPA) showed 4 copies of SNCA (Fig. 1B). Comparative genomic hybridization (Nimblegen 135 K array Roche-Niblegen) showed copy number gain of 0.928 Mb, containing 5 genes including SNCA (Fig. 1C). Single-nucleotide polymorphism (SNP) array showed this to be homozygous. Fluorescent in situ-hybridization demonstrated duplication of SNCA on both alleles (Supplemental Figure 1). The proband's mother's MLPA showed SNCA duplication. Considering the proband's homozygosity for SNCA duplication, her father is likely to have had an SNCA duplication. Less likely, de novo duplication (in addition to duplication inherited from mother) occurred in the proband. SNCA multiplication is a rare cause of autosomal dominant PD.4,5 SNCA triplications (4 copies of SNCA) are fully penetrant; with early-onset and rapidly progressive parkinsonism associated with dementia, autonomic dysfunction, and psychiatric features.4 With SNCA duplications (3 SNCA copies), the disease clinically resembles idiopathic PD5 and reduced penetrance is recognized.6 Our patient has 4 SNCA copies and her clinical phenotype closely matches that of patients with SNCA triplications4 and is in keeping with the single previously described case of SNCA double duplication.7 Homozygous SNCA duplications are a rare cause of young-onset parkinsonism and should be considered in the patients with a family history compatible with recessive inheritance. Early cognitive dysfunction and depression are consistently reported in patients with 4 SNCA copies and may be regarded as “red flags” for SNCA multiplications.