Untranslated Region Polymorphism Research Articles

Human leukocyte antigen-G 3′ untranslated region polymorphism +3142G/C (rs1063320) and haplotypes are associated with manifestations of the American Tegumentary Leishmaniasis in a Northeastern Brazilian population

While the role of cytokine genes has been well documented in the context of Leishmania (Viannia) braziliensis infection, no studies have addressed the influence of human leukocyte antigen-G (HLA-G) in susceptibility/resistance to American Tegumentary Leishmaniasis (ATL). Here, we evaluated the influences of HLA-G, IL-10, TNF-A and IFN-G in the susceptibility and clinical manifestations of ATL.DNA of 114 ATL patients and 346 healthy individuals were sequenced for well-documented polymorphisms in HLA-G 3′ untranslated region (UTR), in IL-10 and TNF-A promoters and in IFN-G intron 1. Soluble HLA-G (sHLA-G) and cytokine levels were evaluated by ELISA and flow cytometry, respectively. Analyses were performed using GraphPad and R-package software.Individuals bearing HLA-G +3142G/G showed an association with increased risk for ATL, whereas those carrying the HLA-G +3142C/G and one copy of UTR6 haplotype, showed an association with decreased risk for ATL. sHLA-G was overexpressed in “susceptible” patients compared to the “resistant’’ one, and also in patients bearing +3142G/G genotype.From these results, HLA-G +3142G/G may be considered as genotype of susceptibility and UTR6 as marker of protection to ATL. Our findings showed a participation of HLA-G in the pathogenesis of the ATL.

Relationship between single nucleotide polymorphisms in the 3'UTR of amyloid precursor protein and risk of Alzheimer's disease and its mechanism.

Background and objective: Deregulation of the expression of amyloid precursor protein (APP) can lead to the development of Alzheimer’s disease (AD). Recent studies have shown that many single nucleotide polymorphisms (SNPs) in the 3′ untranslated region (UTR) of APP are associated with the development of AD. Since microRNAs (miRNAs) are involved in the regulation of APP expression, we believe that the APP 3′UTR polymorphism may affect the regulation of APP expression in miRNAs. Results: The levels of miR-101-3p, miR-153-3p, miR-144-3p, miR-381-3p, and miR-383-5p in plasma of patients with AD were significantly lower than those in the control group. The APP-534G/A site A allele was a protective factor for AD risk (adjusted odds ratio (OR) = 0.700, 95% confidence interval (95% CI): 0.573–0.840, P<0.001). The APP-369C/G site variation was not associated with AD risk. The APP-118C/A site A allele was a protective factor for AD (adjusted OR = 0.762, 95% CI: 0.639–0.897, P=0.001). The APP-534G/A site mutation affects the regulation of APP protein expression by miR-101-3p, miR-144-3p, miR-153-3p, and miR-381-3p, and the mutation of the APP-118C/A site affects miR-101-3p, miR-144-3p, miR-153-3p, and miR-383-5p regulation of APP expression. Conclusion: APP 3′UTR polymorphisms can affect the regulation of APP expression by miRNAs and thus affect the occurrence of AD.

3′UTR variants of TNS3, PHLDB1, NTN4, and GNG2 genes are associated with IgA nephropathy risk in Chinese Han population

BackgroundImmunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis and is characterized by mesangial cell proliferation and agglomeration of the mesangial matrix. MethodsIn this study, we aimed to explore the role of TNS3, PHLDB1, NTN4, and GNG2 3′untranslated region (3′UTR) polymorphisms with the risk of IgAN in a Chinese Han cohort. A logistic recession model was used to calculate the effects of candidate single nucleotide polymorphism (SNP) on IgAN risk after adjusting age and gender difference. In silico prediction was conducted to identify potential functions of SNPs. ResultsThe analysis revealed a significant relationship between the homozygotic genotype for NTN4 rs1362970 A/A and higher risk of IgAN (p = 0.003). Statistically significant associations were found when the sample was stratified by gender and Lee's grade. As a result, NTN4 rs1362970 A/A and GNG2 rs3204008 G/G genotypes were associated with enhanced IgAN risk in males (p = 0.006, p = 0.023, respectively), and the association between the PHLDB1 rs7389 G/T genotype and higher IgAN risk was found in females (p = 0.008). In the Lee's grade III–V subgroup, the rs1369270 in NTN4 was significantly correlated with the risk of IgAN (p = 0.004). Bioinformatics prediction suggested that rs1362970 within NTN4 3′UTR was located in the potential target sequence of miR-483-5p. ConclusionsOur research confirmed that NTN4, GNG2, and PHLDB1 gene polymorphisms were implicated in IgAN susceptibility in Chinese Han population. Further research should be conducted to investigate and validate the mechanism by which the above-mentioned polymorphisms affect IgAN.

CDK6 3'UTR polymorphisms alter the susceptibility to cervical cancer among Uyghur females.

AimsCyclin dependent kinase 6 (CDK6) plays a crucial role in malignant tumor whereas less is reported in cervical cancer development. The aim of this study was to evaluate the effects of CDK6 3' untranslated region (3'UTR) polymorphisms on cervical cancer susceptibility among Uyghur females.MethodsThe genotypes of the six CDK6 variants (rs8179, rs42032, rs42033, rs42034, rs42035, and rs42038) were identified among 306 cervical cancer cases and 310 healthy controls with the Agena MassARRAY platform. The associations of the candidate single nucleotide polymorphisms (SNPs) with the cervical cancer risk were evaluated under genetic models using conditional logistic regression analysis. Bioinformatics analysis was performed for SNP function prediction with the online databases. The expression differences between tumor tissues and normal cervix samples were also examined by Real‐time PCR.Results CDK6 rs8179 and rs42033 were correlated to the decreased risk of cervical cancer in Uyghurs under the allele model (rs8179 and rs42033: OR = 0.60, 95% CI: 0.37–0.99, p = 0.043) and log‐additive model (rs8179 and rs42033: OR = 0.62, 95% CI: 0.38–1.00, p = 0.047). Rs8179, rs42032, and rs42033 were associated with susceptibility to high‐grade cervical cancer in different genetic models as well (p < 0.05). Dataset‐based analysis also uncovered the potential effects of these significant SNPs. In addition, aberrant expression of CDK6 were detected in cervical tumors.ConclusionsOur results suggested the relationships between CDK6 3'UTR polymorphisms and cervical cancer pathogenesis, and the involvement of CDK6 in cervical cancer development among Uyghur females.

The association between IGF1 Gene 3'-UTR polymorphisms and cancer risk: A Meta-analysis.

Insulin-like growth factor 1 (IGF1) gene three prime untranslated region (3'-UTR) polymorphisms have been reported to be associated with cancer risk. However, the conclusions of the relevant studies are not consistent. The present meta-analysis evaluates the relationship between IGF1 gene 3'-UTR polymorphisms (rs5742714, rs6214, and rs6220) and cancer risk. Articles regarding the relationship between IGF1 rs5742714, rs6214, and rs6220 polymorphisms and cancer risk were selected by searching the PubMed, Embase, and Web of Science databases before April 30, 2018. Altogether, we obtained 34 case-controlled studies from 20 articles, including 21,568 cases and 31,199 controls. The strength of associations was quantified using odds ratios (ORs) and the corresponding 95% confidence intervals (CIs). In the present meta-analysis, no significant associations were detected between rs5742714, rs6214, and rs6220 and overall cancer risk. Thus, in stratified analyses, we found that rs6214 was associated with a significantly reduced risk of breast cancer under the allele, heterozygote, and dominant models (A vs G: OR, 0.94, 95% CI,0.88-1.00, P = .044; GA vs GG: OR, 0.88, 95% CI, 0.80-0.97, P = .012; AA + GA vs GG: OR, 0.89, 95% CI, 0.81-0.97, P = .011), as well as pancreatic cancer under the recessive model (AA vs GA + GG: OR, 0.68, 95% CI,0.53-0.87, P = .003). Also, rs6220 was associated with a significantly increased risk of breast cancer under the homozygote model (GG vs AA: OR, 1.23, 95% CI, 1.02-1.48, P = .031). In addition, rs6220 was found to increase overall cancer risk among Caucasians under the allele model (G vs A: OR, 1.06, 95% CI, 1.00-1.13, P = .043). In this meta-analysis, we investigated and reviewed the relationship between IGF1 gene 3'-UTR polymorphisms (rs5742714, rs6214, and rs6220) and cancer risk based on present epidemiological studies. Further studies are needed to draw more precise conclusions in the future.

Germline polymorphisms (SNPs) to predict toxicity and efficacy in FLOT-treated patients with locally advanced gastroesophageal junction or gastric adenocarcinoma—data from the NeoFLOT study

Background: Perioperative treatment is standard of care in Western Europe for locally advanced gastroesophageal cancer (GEC) [gastric and gastroesophageal junction (GEJ) adenocarcinoma]. Predictive markers for toxicity and efficacy prior to start with chemotherapy are desirable, particularly when applying a prolonged neoadjuvant treatment. Here we analyse the impact of previously published SNPs involved in drug metabolism and DNA repair. Methods: Genomic DNA was isolated from 48 tumor samples of patients treated within the NeoFLOT-study. In this trial, FLOT (5-FU, leucovorin, oxaliplatin, docetaxel) was administered every two weeks for 6 cycles chemotherapy prior to surgery. Direct DNA sequencing was carried out for rs25487 [Excision Repair Cross-Complementation Group 1 (XRCC1)], rs1805087 [5-methyltetrahydrofolate-homocysteine methyltransferase (MTR)], rs11615 and rs3212986 (both ERCC1), rs1799793 and rs13181 (both ERCC2), rs1801019 [Orotate Phosphoribosyl Transferase (OPRT)] and rs16430del [thymidylate synthase: 6-bp-deletion in the in the 3' untranslated regulatory region (TS3utrdel)]. Furthermore, thymidylate synthase: 28-bp-tandem repeats in the 5' untranslated region (TS5utr tandem repeat) polymorphism was analysed by Restriction Fragment Length Polymorphism (RFLA). Toxicity was stated according to NCI-CTC version 4.0. For efficacy analysis overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and recurrence-free survival (RFS) were used. Results: ERCC2 rs1799793 was significantly associated with thrombocytopenia ≥ grade 3 (P=0.04) and overall hematotoxicity ≥ grade 3 (P=0.04) while rs13181 was significantly associated with leukopenia ≥ grade 3 (P=0.03). Rs11615 of ERCC1 had a predictive value for leukopenia (P=0.04) and thrombocytopenia (P=0.008). Rs1805087 of MTR and TS5utr tandem repeat polymorphism were both associated with anemia (P=0.04 and 0.04, respectively). Concerning non-hematological toxicity, rs1805087 of MTR was associated with diarrhea (P=0.004). Regarding treatment efficacy, patients harbouring the minor allele of C of OPRT rs1801019 obtained a higher ORR and showed a better but statically non-significant prolonged PFS and OS while rs3212986 of ERCC1 was associated with pCR rate (P=0.006). Conclusions: For single nucleotide polymorphism (SNPs) of ERCC1/2, MTR and TS tandem repeat we could demonstrate the predictive value for toxicity of FLOT in GEC. Furthermore, OPRT rs1801019 might be useful to predict response for neoadjuvant treatment.

Diversity and characterisation of polymorphic 3' untranslated region haplotypes of MICA and MICB genes.

MICA and MICB genes encode ligands that interact with the natural killer (NK) cell activating receptor, NKG2D. These ligands display a highly polymorphic allelic repertoire, although the true functional significance of this polymorphism remains elusive. We previously reported additional polymorphism in the 5' untranslated region (UTR) proximal promoter region of these genes by sequencing international histocompatibility workshop (IHW) cell line DNA promoter and coding regions. The present study extends this analysis by further characterising the 3'UTR region of the same IHW reference panel to achieve a more complete understanding of MICA and MICB haplotype diversity and possible functional relevance. We found 17 extended MICA haplotypes encompassing the coding region and 3'UTR, including four novel haplotypes identified in IHW cell line DNA. This increased to 21 when also considering the 5'UTR proximal promoter region. Analysis of the MICB 3'UTR revealed two novel sequences in cell lines KLO and WIN designated MICB-UTR8 and UTR9, respectively. A total of 11 MICB haplotypes were identified in this study and five were unique. The present study, characterising MICA/B 3'UTR polymorphism utilising IHW reference cell lines, could be useful for future studies investigating the role of microRNA in post-transcriptional repression of gene expression and for immunotherapy strategies to combat cancer progression.

Cooperative translational control of polymorphic BAFF by NF90 and miR-15a.

Polymorphisms in untranslated regions (UTRs) of disease-associated mRNAs can alter protein production. We recently identified a genetic variant in the 3′UTR of the TNFSF13B gene, encoding the cytokine BAFF (B-cell-activating factor), that generates an alternative polyadenylation site yielding a shorter, more actively translated variant, BAFF-var mRNA. Accordingly, individuals bearing the TNFSF13B variant had higher circulating BAFF and elevated risk of developing autoimmune diseases. Here, we investigated the molecular mechanisms controlling the enhanced translation of BAFF-var mRNA. We identified nuclear factor 90 (NF90, also known as ILF3) as an RNA-binding protein that bound preferentially the wild-type (BAFF-WT mRNA) but not BAFF-var mRNA in human monocytic leukemia THP-1 cells. NF90 selectively suppressed BAFF translation by recruiting miR-15a to the 3′UTR of BAFF-WT mRNA. Our results uncover a paradigm whereby an autoimmunity-causing BAFF polymorphism prevents NF90-mediated recruitment of microRNAs to suppress BAFF translation, raising the levels of disease-associated BAFF.

The Role of Complement Activating Collectins and Associated Serine Proteases in Patients With Hematological Malignancies, Receiving High-Dose Chemotherapy, and Autologous Hematopoietic Stem Cell Transplantations (Auto-HSCT).

We conducted a prospective study of 312 patients (194 with multiple myeloma, 118 with lymphomas) receiving high-dose conditioning chemotherapy and autologous hematopoietic stem cell transplantation (auto-HSCT). Polymorphisms of MBL2 and MASP2 genes were investigated and serial measurements of serum concentrations of mannose-binding lectin (MBL), CL-LK collectin and MASP-2 as well as activities of MBL-MASP-1 and MBL-MASP-2 complex were made. Serum samples were taken before conditioning chemotherapy, before HSCT and once weekly after (totally 4-5 samples); in minority of subjects also 1 and/or 3 months post transplantation. The results were compared with data from 267 healthy controls and analyzed in relation to clinical data to explore possible associations with cancer and with chemotherapy-induced medical complications. We found a higher frequency of MBL deficiency-associated genotypes (LXA/O or O/O) among multiple myeloma patients compared with controls. It was however not associated with hospital infections or post-HSCT recovery of leukocytes, but seemed to be associated with the most severe infections during follow-up. Paradoxically, high MBL serum levels were a risk factor for prolonged fever and some infections. The first possible association of MBL2 gene 3′-untranslated region polymorphism with cancer (lymphoma) in Caucasians was noted. Heterozygosity for MASP2 gene +359 A>G mutation was relatively frequent in lymphoma patients who experienced bacteremia during hospital stay. The median concentration of CL-LK was higher in myeloma patients compared with healthy subjects. Chemotherapy induced marked increases in serum MBL and MASP-2 concentrations, prolonged for several weeks and relatively slighter decline in CL-LK level within 1 week. Conflicting findings on the influence of MBL on infections following chemotherapy of myeloma and lymphoma have been reported. Here we found no evidence for an association between MBL deficiency and infection during the short period of neutropenia following conditioning treatment before HSCT. However, we noted a possible protective effect of MBL during follow-up, and suspected that to be fully effective when able to act in combination with phagocytic cells after their recovery.

Human leukocyte antigen-G 3’ untranslated region polymorphisms are associated with asthma severity

Asthma is a genetically complex chronic inflammatory airway disorder, and according to disease pathogenesis, clinical manifestations may vary according to asthma severity. A gene region close to the human leukocyte antigen-G (HLA-G) gene was identified as an independent susceptibility marker for asthma. Considering that the HLA-G immune checkpoint molecule may modulate inflammation, we evaluated the diversity of the HLA-G 3′ untranslated region (3′UTR) in asthmatic patients stratified according to disease severity. We evaluate the entire HLA-G 3′UTR segment in 115 Brazilian patients stratified into mild (n=29), moderate (n=21) and severe asthmatics (n=65), and in 116 healthy individuals. HLA-G 3′UTR typing was performed using Sanger sequencing. The multiple comparisons among patients stratified according to disease severity revealed several associations; however, after Bonferroni’s correction, the following results remained significant: i) the +3010C and +3142G alleles were overrepresented in mild asthma patients when compared to controls; ii) the +3010G and +3142C alleles were overrepresented in severe asthma patients in comparison to patients with mild asthma. In conclusion, the +3010C/G and +3142C/G HLA-G 3′UTR variation sites were differentially associated according to asthma severity.

Programmed cell death-1 3'-untranslated region polymorphism is associated with spontaneous clearance of hepatitis B virus infection.

Hepatitis B virus (HBV)-specific CD8+ T cells play an important role in the clearance of HBV infection. Programmed cell death-1 (PD-1), an immunosuppressive molecule that regulates T-cell activation and peripheral immune tolerance, is increasingly shown to influence the outcome of HBV infection. rs10204525, a single-nucleotide polymorphism in the 3'-untranslated region (3'-UTR) of PD-1, has been associated with susceptibility and disease progression of chronic HBV infection in far-eastern patients. The aim of our study was to assess the impact of rs10204525 variation on HBV infection in Moroccan patients. A total of 236 patients with chronic HBV infection and 134 individuals with spontaneous HBV resolution were genotyped using a Taqman assay. In addition, PD-1 mRNA expression in peripheral blood nuclear cells was determined by quantitativereverse-transcription polymerase chain reaction. We found that the AA genotype is protective (odds ratio, 0.43; 95% confidence interval, 0.19 to 0.97; P = 0.038) against HBV infection. Interestingly, PD-1 messenger RNA (mRNA) expression analysis has revealed that chronic HBV carriers with GG and GA displayed higher levels of PD-1 mRNA compared with corresponding genotypes in resolved subjects (P = 0.031 and0.014, respectively). Our data suggest that Mediterranean HBV-infected patients carrying PD-1 GG and GA genotypes at rs10204525 have high PD-1 mRNA expression and may be more prone to installation of chronicity.

Genotype-based selection of treatment of patients with advanced colorectal cancer (SETICC): a pharmacogenetic-based randomized phase II trial

There has been little progress toward personalized therapy for patients with metastatic colorectal cancer (mCRC). TYMS-3' untranslated region (UTR) 6 bp ins/del and ERCC1-118C/T polymorphisms were previously reported to facilitate selecting patients for fluoropyrimidine-based treatment in combination with oxaliplatin as first-line therapy. We assessed the utility of these markers in selecting therapy for patients with mCRC. This randomized, open-label phase II trial compared bevacizumab plus XELOX (control) versus treatment tailored according to TYMS-3'UTR 6 bp ins/del and ERCC1-118C/T polymorphisms. Patients randomized to the experimental treatment received bevacizumab plus FUOX, FUIRI, XELIRI, or XELOX depending on their combination of favorable polymorphisms for FUOX treatment (TYMS-3'UTR ins/del or del/del; ERCC1-118T/T). Progression-free survival (PFS) was the primary end point. Overall, 195 patients were randomized (control n = 65; experimental n = 130). The primary objective was not met: median PFS was 9.4 months in the control group and 10.1 months in the experimental group (P = 0.745). Median overall survival was similar in both groups (16.5 versus 19.1 months, respectively; P = 0.797). Patients in the experimental group had a significantly higher overall response rate (ORR; 65% versus 47% in the control group; P = 0.042) and R0 resection rate (86% versus 44%, respectively; P = 0.018). Neuropathy, hand-foot syndrome, thrombocytopenia, and dysesthesia were significantly less common in the experimental group. This study did not show survival benefits after treatment personalization based on polymorphisms in mCRC. However, the improved ORR and R0 resection rate and fewer disabling toxicities suggest that tailoring therapy by TYMS-3'UTR and ERCC1-118 polymorphisms warrants further investigation in patients with mCRC. NCT01071655.

Cooperative Translational Control of Polymorphic BAFF by NF90 and miR-15a

Polymorphisms in untranslated regions (UTRs) of disease-associated mRNAs can alter protein production. We recently identified a genetic variant in the 3’UTR of the TNFSF13B gene, encoding the cytokine BAFF (B-cell-activating factor), that generates an alternative polyadenylation site yielding a shorter, more actively translated variant, BAFF-var mRNA. Accordingly, individuals bearing the TNFSF13B variant had higher circulating BAFF and elevated risk of developing autoimmune diseases. Here, we investigated the molecular mechanisms controlling the enhanced translation of BAFF-var mRNA. We identified nuclear factor 90 (NF90, also known as ILF3) as an RNA-binding protein that bound preferentially the wild-type (BAFF-WT mRNA) but not BAFF-var mRNA in human monocytic leukemia THP-1 cells. NF90 selectively suppressed BAFF translation by recruiting miR-15a to the 3’UTR of BAFF-WT mRNA. Our results uncover a paradigm whereby an autoimmunity-causing BAFF polymorphism prevents NF90-mediated recruitment of microRNAs to suppress BAFF translation, raising the levels of disease-associated BAFF.

A 3' untranslated region polymorphism rs2304277 in the DNA repair pathway gene OGG1 is a novel risk modulator for urothelial bladder carcinoma.

Altered DNA repair capacity may affect an individual's susceptibility to cancers due to compromised genomic integrity. This study was designed to elucidate the association of selected polymorphisms in DNA repair genes with urothelial bladder carcinoma (UBC). OGG1 rs1052133 and rs2304277, XRCC1 rs1799782 and rs25487, XRCC3 rs861539, XPC rs2228001, and XPD rs13181 were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 200 UBC cases and 200 controls. We found association of OGG1 rs2304277 [odds ratio (OR)GG = 3.55, 95% confidence interval (CI) = 1.79-7.06] and XPC rs2228001 (ORAC = 2.38, 95% CI = 1.43-3.94) with UBC. In stratified analysis with respect to smoking status, OGG1 rs2304277 and XPC rs2228001 exhibited increased risk in smokers [(rs2304277 ORGG = 4.96, 95% CI = 1.51-16.30) (rs2228001 ORAC = 2.19, 95% CI = 1.02-4.72)] as well as nonsmokers [(rs2304277 ORGG = 2.95, 95% CI = 1.26-6.90) (rs2228001 ORAC = 2.57, 95% CI = 1.31-5.04)]. These polymorphisms were also associated with both low-grade [(rs2304277 ORGG = 3.73, 95% CI = 1.72-8.09) (rs2228001 ORAC = 2.18, 95% CI = 1.21-3.92)] and high-grade tumors [(rs2304277 ORGG = 3.45, 95% CI = 1.52-7.80) (rs2228001 ORAC = 2.81, 95% CI = 1.48-5.33)] as well as with non-muscle-invasive bladder cancer [(rs2304277 ORGG = 4.03, 95% CI = 1.87-8.67) (rs2228001 ORAC = 2.14, 95% CI = 1.20-3.81)] and muscle-invasive bladder cancer [(rs2304277 ORGG = 3.06, 95%CI = 1.31-7.13) (rs2228001 ORAC = 2.95, 95%CI = 1.51-5.75)]. This is the first study on DNA repair gene polymorphisms and UBC in the Pakistani population. It identifies OGG1 rs2304277 and replicates XPC rs2228001 as significant modulators of UBC susceptibility.

Association of a 3' untranslated region polymorphism in proprotein convertase subtilisin/kexin type 9 with HIV viral load and CD4+ levels in HIV/hepatitis C virus coinfected women.

To assess variation in genes that regulate cholesterol metabolism in relation to the natural history of HIV infection. Cross-sectional and longitudinal analysis of the Women's Interagency HIV Study. We examined 2050 single nucleotide polymorphisms (SNPs) in 19 genes known to regulate cholesterol metabolism in relation to HIV viral load and CD4 T-cell levels in a multiracial cohort of 1066 antiretroviral therapy-naive women. Six SNPs were associated with both HIV viral load and CD4 T-cell levels at a false discovery rate of 0.01. Bioinformatics tools did not predict functional activity for five SNPs, located in introns of nuclear receptor corepressor 2, retinoid X receptor alpha (RXRA), and tetratricopeptide repeat domain 39B. Rs17111557 located in the 3' untranslated region of proprotein convertase subtilisin/kexin type 9 (PCSK9) putatively affects binding of hsa-miR-548t-5p and hsa-miR-4796-3p, which could regulate PCSK9 expression levels. Interrogation of rs17111557 revealed stronger associations in the subset of women with HIV/hepatitis C virus (HCV) coinfection (n = 408, 38% of women). Rs17111557 was also associated with low-density lipoprotein cholesterol levels in HIV/HCV coinfected (β: -10.4; 95% confidence interval: -17.9, -2.9; P = 0.007), but not in HIV monoinfected (β:1.2; 95% confidence interval: -6.3, 8.6; P = 0.76) women in adjusted analysis. PCSK9 polymorphism may affect HIV pathogenesis, particularly in HIV/HCV coinfected women. A likely mechanism for this effect is PCSK9-mediated regulation of cholesterol metabolism. Replication in independent cohorts is needed to clarify the generalizability of the observed associations.

P071 The reference: Sequence-level comparison of HLA loci in unrelated hematopietic cell transplant (HCT) pairs

Aim HLA matching by allele-level genotyping is largely based on genetic similarity between exons which encode the antigen recognition site (ARS). Next-generation sequencing can identify polymorphisms in non-ARS exons, introns, and untranslated regions, but the impact on HCT outcome is unclear. Here we report a bioinformatics approach to directly compare recipient and donor HLA sequences and identify regions of genetic dissimilarity. Methods The Illumina TruSight HLA NGS assay was used to sequence the HLA-A, -B, -C, DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 loci for 169 paired donor and recipient samples obtained from the CIBMTR repository. Assign v2.1 software was used to generate HLA genotypes and consensus sequences from each locus. Recipient consensus sequences are aligned using Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo ) to both the corresponding reference allele in IMGT/HLA v3.27.0 and the donor consensus sequence for that locus. Mismatches between consensus sequences are annotated according to genetic features (exon, intron, UTR) obtained from the IMGT/HLA reference allele. Indels are considered when adjusting genetic feature locations. Exonic sequence alignments and amino acid substitutions are then confirmed by comparison with IMGT/HLA. Alignments are visualized using custom software to highlight nucleotide and amino acid differences and provide summary statistics. Results The algorithm was validated using HCT pairs with known allele-level mismatches to confirm correct assignment of ARS differences. The algorithm accurately identifies and categorizes HCT pairs with HLA loci that differ outside the ARS region as well as those that do not. In addition, the algorithm discovers instances where the consensus phase genotype is ambiguous. Conclusions We report a novel bioinformatics approach to directly compare donor and recipient consensus HLA sequences to identify and visualize regions of sequence heterogeneity. Furthermore, our automated pipeline aids in the discovery of novel “feature-specific” mismatch patterns, offering an additional means of comparison beyond traditional HLA allele assignment. We plan to utilize this approach to categorize HCT pairs for analysis of clinical outcome measures by matching status at each region of interest.

Relationship between single-nucleotide polymorphisms in un-translated region of human leukocyte antigen-G and preeclampsia

Purpose: The aim of this study was to unravel the link between human leukocyte antigen-G untranslated region (HLA-G 3-UTR) single-nucleotide polymorphism (SNP) and preeclampsia by examining polymorphisms in HLA-G 3-UTR in preeclampsia patients and their newborns, as well as those of women with normal pregnancy and their newborns.Methods: Single pregnant mothers and their newborns at the Third Affiliated Hospital of Zhengzhou University, China, were divided into preeclampsia group (144 cases), and normal pregnancy group (122 cases). Blood samples from the two groups were collected for DNA extraction, and the DNA samples were analyzed for HLA-G 3'-UTR SNP by polymerase chain reaction (RT-PCR) and direct sequencing method.Results: The frequency of CG genotype in HLA-G 3-UTR 3127 gene in the preeclampsia group was significantly lower, while the frequency of AA genotype in HLA-G 3-UTR 3172 gene in the preeclampsia group was significantly higher (p &lt; 0.05) when compared with the normal pregnancy group. Furthermore, frequencies of the compatible genotype AA/AA (mothers/newborns) in HLA-G 3-UTR 3172 gene in the preeclampsia group were significantly higher relative to the normal pregnancy group (p&lt; 0.05).Conclusion: These results suggest that when CC/GG is the compatible genotype in HLA-G 3-UTR 3127 gene of mothers/newborns, preeclampsia risk may decrease, but it may increase if the compatible genotype is AA/AA. Thus, SNP in HLA-G 3-UTR 3127 gene may be a protective factor against preeclampsia in Chinese Han pregnant women, while SNP in 3172 gene may be associated with susceptibility to preeclampsia.Keywords: Single-nucleotide polymorphisms, Human leukocyte antigen-G, Un-translated Region, Preeclampsia

HLA-G 3'UTR Polymorphisms Predict Drug-Induced G3-4 Toxicity Related to Folinic Acid/5-Fluorouracil/Oxaliplatin (FOLFOX4) Chemotherapy in Non-Metastatic Colorectal Cancer.

Polymorphisms in drug-metabolizing enzymes might not completely explain inter-individual differences in toxicity profiles of patients with colorectal cancer (CRC) that receive folinic acid/5-fluorouracil/oxaliplatin (FOLFOX4). Recent data indicate that the immune system could contribute to FOLFOX4 outcomes. In light of the immune inhibitory nature of human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex (MHC) class I molecule, we aimed to identify novel genomic markers of grades 3 and 4 (G3-4) toxicity related to FOLFOX4 therapy in patients with CRC. We retrospectively analyzed data for 144 patients with stages II-III CRC to identify HLA-G 3′ untranslated region (3′UTR) polymorphisms and related haplotypes and evaluate their impact on the risk of developing G3-4 toxicities (i.e., neutropenia, hematological/non-hematological toxicity, neurotoxicity) with logistic regression. The rs1610696-G/G polymorphism was associated with increased risk of G3-4 neutropenia (OR = 3.76, p = 0.015) and neurotoxicity (OR = 8.78, p = 0.016); rs371194629-Ins/Ins was associated with increased risk of neurotoxicity (OR = 5.49, p = 0.027). HLA-G 3′UTR-2, which contains rs1610696-G/G and rs371194629-Ins/Ins polymorphisms, was associated with increased risk of G3-4 neutropenia (OR = 3.92, p = 0.017) and neurotoxicity (OR = 11.29, p = 0.009). A bootstrap analysis confirmed the predictive value of rs1610696 and rs371194629, but the UTR-2 haplotype was validated only for neurotoxicity. This exploratory study identified new HLA-G 3′UTR polymorphisms/haplotypes as potential predictive markers of G3-4 toxicities in CRC.

Analysis of CKM (creatine kinase, muscle) polymorphism frequencies.

In this experiment the prevalence of a 3’ untranslated region (UTR) polymorphism in the creatine kinase, muscle (CKM) gene was explored. Previous studies indicated that AA, AG and GG genotypes were found in 51%, 44% and 5% of the population. CKM is found in skeletal and heart muscle tissue, and its presence in the bloodstream is an indicator of muscle damage. The objective of this study was to replicate prior research utilizing CKM and to assess the frequency in the sample. It was hypothesized that the previous research could be replicated and that the genotype frequencies in the sample would match published research on CKM. Fifty-five individuals (16 male, 39 female) were sampled through the use of a cheek swab, and were worked up using Chelex resin techniques. Genotyping was performed using PCR with previously designed primers followed by digestion with the NcoI. When running results on the final gel, A allele digestion yielded fragments of 63bp and 52bp, while the G allele was undigested, resulting in a 115bp fragment. Results were obtained for 47 individuals and 12 (26%) were AG genotype while 43 (74%) were AA genotype. These results indicated that the genotyping of the 3’ UTR region of CKM was successful, but genotype frequencies were different from those previously documented. Future plans would be to assess the polymorphism frequency on a larger population.

Integrating TYMS, KRAS and BRAF testing in patients with metastatic colorectal cancer.

AIMTo investigate the impact of thymidylate synthase (TYMS), KRAS and BRAF in the survival of metastatic colorectal cancer (mCRC) patients treated with chemotherapy.METHODSClinical data were collected retrospectively from records of consecutive patients with mCRC treated with fluoropyrimidine-based chemotherapy from 1/2005 to 1/2007. Formalin-fixed paraffin-embedded tissues were retrieved for analysis. TYMS genotypes were identified with restriction fragment analysis PCR, while KRAS and BRAF mutation status was evaluated using real-time PCR assays. TYMS gene polymorphisms of each of the 3’ untranslated region (UTR) and 5’UTR were classified into three groups according to the probability they have for high, medium and low TYMS expression (and similar levels of risk) based on evidence from previous studies. Univariate and multivariate survival analyses were performed.RESULTSThe analysis recovered 89 patients with mCRC (46.1% de novo metastatic disease and 53.9% relapsed). Of these, 46 patients (51.7%) had colon cancer and 43 (48.3%) rectal cancer as primary. All patients were treated with fluoropyrimidine-based chemotherapy (5FU or capecitabine) as single-agent or in combination with irinotecan or/and oxaliplatin or/and bevacizumab. With a median follow-up time of 14.8 mo (range 0-119.8), 85 patients (95.5%) experienced disease progression, and 63 deaths (70.8%) were recorded. The 3-year and 5-year OS rate was 25.4% and 7.7% while the 3-year progression-free survival rate was 7.1%. Multivariate analysis of TYMS polymorphisms, KRAS and BRAF with clinicopathological parameters indicated that TYMS 3’UTR polymorphisms are associated with risk for disease progression and death (P < 0.05 and P < 0.03 respectively). When compared to tumors without any del allele (genotypes ins/ins and ins/loss of heterozygosity (LOH) linked with high TYMS expression) tumors with del/del genotype (low expression group) and tumors with ins/del or del/LOH (intermediate expression group) have lower risk for disease progression (HR = 0.432, 95%CI: 0.198-0.946, P < 0.04 and HR = 0.513, 95%CI: 0.287-0.919, P < 0.03 respectively) and death (HR = 0.366, 95%CI: 0.162-0.827, P < 0.02 and HR = 0.559, 95%CI: 0.309-1.113, P < 0.06 respectively). Additionally, KRAS mutation was associated independently with the risk of disease progression (HR = 1.600, 95%CI: 1.011-2.531, P < 0.05). The addition of irinotecan in 1st line chemotherapy was associated independently with lower risk for disease progression and death (HR = 0.600, 95%CI: 0.372-0.969, P < 0.04 and HR = 0.352, 95%CI: 0.164-0.757, P < 0.01 respectively).CONCLUSIONThe TYMS genotypes ins/ins and ins/LOH associate with worst prognosis in mCRC patients under fluoropyrimidine-based chemotherapy. Large prospective studies are needed for validation of our findings.