UTR Haplotypes Research Articles

HLA-G high-expressor 3'UTR markers are linked to gastric cancer development and survival.

Gastric cancer ranks fifth in both world prevalence and lethality, with a 5-year survival of less than 30%. HLA-G, a non-classical class I HLA gene, has emerged as a potential marker for cancer susceptibility and prognosis due to its immunomodulatory properties. Its level of expression is regulated by polymorphisms in the 3' untranslated region (3'UTR) polymorphisms, which form various combined haplotypes (UTR-1 to -9). In this study, we examined HLA-G 3'UTR polymorphisms in paired tissue samples from 111 patients with gastric adenocarcinoma and 119 healthy controls. Polymorphism analysis was performed using PCR and Sanger sequencing, followed by statistical analysis using SNPStats software. Survival analysis was conducted using Kaplan-Meier curves and multivariate Cox regression models. High-expressor HLA-G 3'UTR haplotypes (UTR-1 and UTR-6) were significantly associated with gastric cancer susceptibility, indicating a potential role in tumor immune evasion. Additionally, the 14 base pair insertion/deletion polymorphism (14bp I/D) emerged as a prognostic marker, with D/D genotype carriers showing lower survival rates compared to I/D and I/I genotype carriers. Our study highlights the clinical relevance of HLA-G polymorphisms in gastric cancer, suggesting their potential as prognostic markers and therapeutic targets. Further elucidation of HLA-G-related pathways could lead to personalized treatment strategies and improved patient outcomes in gastric cancer.

Association of HLA-G 3'UTR polymorphisms with hepatitis B virus infection in Tunisian population.

Hepatitis B virus (HBV) infection is a major public health burden. The mechanisms of immune evasion during chronic HBV (CHB) infection are poorly understood. Human leukocyte antigen (HLA)-G, an immune checkpoint molecule, plays a crucial role in the tolerance mechanisms of various infectious diseases. The 3' untranslated region (3'UTR), including the HLA-G + 3142 C > G polymorphism (rs1063320) and the 14-pb Ins/Del (rs66554220) has been strongly suggested to influence HLA-G expression. This study conducted a case-control analysis to evaluate the potential correlation between the HLA-G + 3142 C > G polymorphism and HBV infection outcome in a Tunisian cohort. The HLA-G + 3142 C > G polymorphism was analysed by PCR-RFLP in 242 patients with chronic HBV infection (116 males and 126 females), 241 healthy controls (116 males and 125 females), and 100 spontaneously resolved subjects (52 males and 48 females). Patients with chronic HBV infection showed a higher frequency of the + 3142G allele compared to healthy controls and spontaneously resolved subjects (p = 0.001 and p = 0.002, respectively). An association between the + 3142G allele and high HBV DNA levels was observed when HBV patients were stratified based on their HBV DNA levels (p = 0.016). Furthermore, the dominant model (GG + GC vs CC) was associated with liver function parameters, including AST, ALT, and high HBV DNA levels (p = 0.04, p < 0.001 and p = 0.002, respectively). However, there was no significant association found between this polymorphism and the fibrosis stage (p = 0.32). The haplotype analysis, using a subset of previously published data on the HLA-G 14-pb Ins/Del polymorphism, revealed an association between the Ins/G haplotype and chronic HBV infection (H1: InsG, p < 0.001). Our findings suggest that the + 3142G allele is a risk factor for the persistence and progression of HBV infection, while the + 3142C allele serves as a protective allele associated with the spontaneous resolution of the infection. Additionally, the HLA-G 3'UTR haplotype Ins/G is associated with chronic HBV infection in the Tunisian population.

HLA-G 3'UTR haplotype analyses in HCV infection and HCV-derived cirrhosis, hepatocellular carcinoma and fibrosis.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Chronic HCV infection is also an important cause of hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV has the capacity to evade immune surveillance by altering the host immune response. Moreover, variations in immune-related genes can lead to differential susceptibility to HCV infection as well as interfere on the susceptibility to the development of hepatic fibrosis, cirrhosis and HCC. The human leucocyte antigen G (HLA-G) gene codes for an immunomodulatory protein known to be expressed in the maternal-foetal interface and in immune-privileged tissues. The HLA-G 3' untranslated region (3'UTR) is important for mRNA stability, and variants in this region are known to impact gene expression. Studies, mainly focusing in a 14bp insertion/deletion polymorphism, have correlated HLA-G 3'UTR with susceptibility to viral infections, but other polymorphic variants in the HLA-G 3'UTR might also affect HCV infection as they are inherited as haplotypes. The present study evaluated HLA-G 3'UTR polymorphisms and performed linkage disequilibrium test and haplotype assembly in 286 HCV infected patients who have developed fibrosis, cirrhosis or HCC, as well as in 129 healthy control subjects. Haplotypes UTR-1, UTR-2 and UTR-3 were the most observed in HCV+ patients, in the frequencies of 0.276, 0.255 and 0.121, respectively. No statistically significant difference was observed between HCV+ and control subjects, even when patients were grouped according to outcome (HCC, cirrhosis or fibrosis). Despite that, some trends in the results were observed, and therefore, we cannot rule out the possibility that variants associated to high HLA-G expression can be involved in HCV infection susceptibility.

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients.

Triple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets. To follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3' UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival. sHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3' UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy response. The results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets.

Primary age-related tauopathy in a Finnish population-based study of the oldest old (Vantaa 85+).

AimsFew studies have investigated primary age‐related tauopathy (PART) in a population‐based setting. Here, we assessed its prevalence, genetic background, comorbidities and features of cognitive decline in an unselected elderly population.MethodsThe population‐based Vantaa 85+ study includes all 601 inhabitants of Vantaa aged ≥ 85 years in 1991. Neuropathological assessment was possible in 301. Dementia (DSM IIIR criteria) and Mini‐Mental State Examination (MMSE) scores were assessed at the baseline of the study and follow‐ups. PART subjects were identified according to the criteria by Crary et al and were compared with subjects with mild and severe Alzheimer's disease (AD) neuropathological changes. The effects of other neuropathologies were taken into account using multivariate and sensitivity assays. Genetic analyses included APOE genotypes and 29 polymorphisms of the MAPT 3′ untranslated region (3′UTR region).ResultsThe frequency of PART was 20% (n = 61/301, definite PART 5%). When PART subjects were compared with those with severe AD pathology, dementia was less common, its age at onset was higher and duration shorter. No such differences were seen when compared with those with milder AD pathology. However, both AD groups showed a steeper decline in MMSE scores in follow‐ups compared with PART. APOE ε4 frequency was lower, and APOE ε2 frequency higher in the PART group compared with each AD group. The detected nominally significant associations between PART and two MAPT 3′UTR polymorphisms and haplotypes did not survive Bonferroni correction.ConclusionsPART is common among very elderly. PART subjects differ from individuals with AD‐type changes in the pattern of cognitive decline, associated genetic and neuropathological features.

Association of HLA-G 3′UTR polymorphisms and haplotypes with colorectal cancer susceptibility and prognosis

Human leukocyte antigen (HLA)-G has been considered as an immune modulator in several types of cancers. Its genetic polymorphisms may potentially affect the risk of developing colorectal cancer (CRC). The overall purpose of this study was to analyze the implication of HLA-G 3′untranslated region (3′UTR) polymorphisms particularly 14 pb insertion/deletion (Ins/Del; rs371194629) and + 3142C/G (rs1063320) in CRC susceptibility and progression.A comparative analysis between patients (N = 233) and controls (N = 241) demonstrated that Del allele (Odds Ratios (OR) = 1.41, 95% CI = 1.091–1.819, p = 0.008), the homozygous Del/Del genotype (OR = 1.80, 95% CI = 1.205–2.664, p = 0.003) and the codominant C/G genotype (OR = 1.59, 95% CI = 1.106–2.272, p = 0.013) were associated to CRC risk. As expected, the DelG haplotype was associated with CRC susceptibility (OR = 1.47, 95% CI = 1.068–2.012, p = 0.018). Assessment of patients' survival by Kaplan-Meier analysis indicated that the Del allele and the homozygous Del/Del genotype were associated with reduced event free survival (EFS) (Respectively, p = 0.009 and p = 0.05). Interestingly, the Del allele and the homozygous Del/Del genotype have been revealed as independent prognostic factors for poor EFS in patients with CRC. Additionally, haplotypes analysis revealed that DelG haplotype was linked with significant increase in CRC risk (log-rank; EFS: p = 0.02). Inversely, the InsC haplotype was associated with a significant reduced CRC risk (log-rank; Overall survival (OS): p < 10–6; EFS: p = 0.01). Multivariate Cox regression analysis revealed that the InsC haplotype was independently associated with significantly longer EFS (p = 0.021, HR = 0.636, 95% CI = 0.433–0.935).These findings support the implication of HLA-G polymorphisms in the CRC susceptibility suggesting HLA-G as a potent prognostic and predictive indicator for CRC. Insight into mechanisms underlying HLA-G polymorphisms could allow for the development of targeted care for CRC patients according to their genetic profile.

Examining extended human leukocyte antigen-G and HLA-F haplotypes: the HLA-G UTR-4 haplotype is associated with shorter time to pregnancy in an infertility treatment setting when both female and male partners are carriers

To study the impact of extended human leukocyte antigen (HLA)-G and HLA-F haplotypes on time to pregnancy as measured by the number of treatment cycles in a cohort of couples in infertility treatment. Prospective cohort study of couples undergoing infertility treatment. University hospital. A cohort of 127 couples and four single women in infertility treatment. Next-generation sequencing of the HLA-G gene and genotyping of three HLA-F locus single-nucleotide polymorphisms (SNPs). Extended HLA-F.HLA-G haplotypes, HLA-G promoter haplotypes and HLA-G 3'UTR haplotypes and their association with time to pregnancy as measured by number of treatment cycles until achievement of pregnancy with a live birth. Linkage disequilibrium between HLA-G variations and three HLA-F locus SNPs that impact time to pregnancy. The effect of the HLA-G 3'UTR haplotype, UTR-4, was significantly increased, or modified, if the partner was a carrier compared to being a noncarrier. Extended HLA-F.HLA-G haplotypes, HLA-G promoter haplotypes, and the HLA-G 14 bp indel of the female partners were not associated with time to pregnancy. However, a trend for an association of the HLA-G 14bp insertion allele with a higher frequency of miscarriage than the 14bp deletion allele was observed. Certain HLA-G variations are in linkage disequilibrium with three HLA-F locus SNPs that influence time to pregnancy. HLA-G UTR-4 is significantly associated with time to pregnancy in couples undergoing infertility treatment. The findings could imply that both male and female HLA class Ib genetics have clinical relevance in reproduction.

HLA-G whole gene amplification reveals linkage disequilibrium between the HLA-G 3'UTR and coding sequence.

Polymorphic sites in the HLA‐G gene may influence expression and function of the protein. Knowledge of the association between high‐resolution HLA‐G alleles and 3‐prime untranslated (3′UTR) haplotypes is useful for studies on the role of HLA‐G in transplantation, pregnancy, and cancer. We developed a next generation sequencing (NGS)‐based typing assay enabling full phasing over the whole HLA‐G gene sequence with inclusion of the 3′UTR region. DNA from 171 mother‐child pairs (342 samples) was studied for: (a) HLA‐G allele information by the NGSgo‐AmpX HLA‐G assay, (b) 3′UTR haplotype information by an in‐house developed sequence‐based typing method of a 699/713 base pair region in the 3′UTR, and (c) the full phase HLA‐G gene sequence, by combining primers from both assays. The mother to child inheritance allowed internal verification of newly identified alleles and of association between coding and UTR regions. The NGSgo workflow compatible with Illumina platforms was employed. Data was interpreted using NGSengine software. In 99.4% of all alleles analyzed, the extended typing was consistent with the separate allele and 3′UTR typing methods. After repeated analysis of four samples that showed discrepancy, consistency reached 100%. A high‐linkage disequilibrium between IPD‐IMGT/HLA Database‐defined HLA‐G alleles and the extended 3′UTR region was identified (D′ = 0.994, P < .0001). Strong associations were found particularly between HLA‐G*01:04 and UTR‐3, between HLA‐G*01:01:03 and UTR‐7, and between HLA‐G*01:03:01 and UTR‐5 (for all: r = 1). Six novel HLA‐G alleles and three novel 3′UTR haplotype variants were identified, of which three and one, respectively, were verified in the offspring.

Full sequence of mutant huntingtin 3'-untranslated region and modulation of its gene regulatory activity by endogenous microRNA.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3'-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3'-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3'-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3'-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3'-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.

Glutathione S-transferase theta genotypes and environmental exposures in the risk of canine transitional cell carcinoma.

IntroductionTransitional cell carcinoma (TCC) in humans is associated with environmental exposures and variants in glutathione S‐transferase (GST) genes. Scottish Terriers have a high breed risk for TCC, but the relationship between genetic and environmental risk in dogs is not fully understood.HypothesesScottish Terriers have a higher frequency of GST‐theta variants compared to lower risk breeds. Dogs with TCC of any breed have a higher frequency of GST‐theta variants along with higher environmental exposures, compared to controls.AnimalsOne hundred and five Scottish Terriers and 68 controls from lower risk breeds; 69 dogs of various breeds with TCC, and 72 breed‐ and sex‐matched unaffected geriatric dogs.MethodsIn this prospective case‐control study, dogs were genotyped for 3 canine GST‐theta variants: GSTT1 I2+28 G>A, a GSTT1 3′UTR haplotype, and GSTT5 Asp129_Gln130del. Owners of dogs with TCC and unaffected geriatric controls completed a household environmental questionnaire.ResultsThe GSTT1 3′UTR haplotype and GSTT5 Asp129_Gln130del variants were significantly underrepresented in Scottish Terriers (minor allele frequency [MAF] = 0.000 for both), compared to dogs from lower risk breeds (MAF = 0.108 and 0.100; P ≤ .0002). Dogs with TCC did not differ from unaffected geriatric controls across the 3 investigated loci. Transitional cell carcinoma was associated with household insecticide use (odds ratio [OR] = 4.28, 95% confidence interval [CI] = 1.44‐12.33, P = .02), and was negatively associated with proximity to a farm (OR = 0.49, 95% CI = 0.25‐0.99, P = .04).Conclusions and Clinical ImportanceLow‐activity GST‐theta loci are unlikely contributors to TCC risk in dogs. Increased risk is associated with household insecticide use, and possibly with less rural households.

HLA-G, -E and -F regulatory and coding region variability and haplotypes in the Beninese Toffin population sample

HLA-G/E/F genes exhibit immunomodulatory properties and are expressed in placenta. Little attention has been devoted to the study of these genes in sub-Saharan African populations, which are yet the most diverse. To fill this gap, we evaluated the complete gene variability, approximately 5.1 kb for HLA-G (n = 149), 7.7 kb for HLA-E (n = 150) and 6.2 kb for HLA-F (n = 152) in the remote Beninese Toffin population, using massive parallel sequencing. Overall, 96, 37 and 68 variable sites were detected along the entire HLA-G, -E and -F, respectively, arranged into region-specific haplotypes; i.e., promoter haplotypes (16, 19, and 15 respectively), coding haplotypes (19, 15, and 29 respectively), 3’ untranslated region (3′UTR) haplotypes (12, 7 and 2, respectively) and extended haplotypes (33, 31 and 32 respectively). All promoter/coding/3’UTR haplotypes followed the patterns already described in worldwide populations. HLA-E was the most conserved, exhibiting mainly two full-length encoded-molecules (E*01:01 and E*01:03), followed by HLA-F, three full-length proteins (F*01:01, F*01:02 and F*01:03) and HLA-G, four proteins: three full-length (G*01:01, G*01:03 and G*01:04) and one truncated (G*01:05N). Although HLA-G/E/F alleles in the Toffin population were the most frequently observed worldwide, the frequencies of the coding haplotypes were closely similar to those described for other African populations (Guinea-Conakry and Burkina-Faso), when compared to non-African ones (Brazilian), indicating that variable sites along these genes were present in Africa before human dispersion.

Evaluation of DEFB1 polymorphisms in individuals with chronic periodontitis and diabetes mellitus type 2 in a population of northeastern Brazil.

The role of genetic variations in genes related to innate response, as β-defensin-1 (DEFB1), in the context of chronic periodontitis (CP) and diabetes mellitus type 2 (DM2), is still not clear. The present study evaluates the distribution of DEFB1 single nucleotide polymorphisms (SNPs) 5'-untranslated (5'UTR) region and its relation with the CP in DM2 individuals in northeastern Brazilians. Two hundred and eighty individuals participated in the study, being 116 DM2+CP, 95 CP, and 69 healthy individuals. Three known DEFB1 functional SNPs [-52 G>A (rs1799946), -44 C>G (rs1800972), -20 G>A (rs11362)] were genotyped with allele-specific assays. Association was found for the DEFB1 -20 G>A SNP. The G allele, the GA and GG genotypes were significantly (P<0.05) more frequent in the DM2+CP (59.5%, 50%, and 34.5%, respectively) and CP (61%, 44.2%, and 38.9%, respectively) than in healthy individuals (26.8%, 36.2%, and 8.7%, respectively). The GCG and ACG combinations (-52, -44, -20) were significantly more frequent among DM2+CP and CP than in the healthy individuals. The results indicate that genetic variations of DEFB1 gene (SNP-20: G allele and GA and GG genotypes) and the DEFB1 5'UTR haplotypes (GCG and ACG) may be associated with a susceptibility to CP in DM2 individuals as well as CP individuals without DM2.

HLA-E regulatory and coding region variability and haplotypes in a Brazilian population sample

The HLA-E gene is characterized by low but wide expression on different tissues. HLA-E is considered a conserved gene, being one of the least polymorphic class I HLA genes. The HLA-E molecule interacts with Natural Killer cell receptors and T lymphocytes receptors, and might activate or inhibit immune responses depending on the peptide associated with HLA-E and with which receptors HLA-E interacts to. Variable sites within the HLA-E regulatory and coding segments may influence the gene function by modifying its expression pattern or encoded molecule, thus, influencing its interaction with receptors and the peptide. Here we propose an approach to evaluate the gene structure, haplotype pattern and the complete HLA-E variability, including regulatory (promoter and 3′UTR) and coding segments (with introns), by using massively parallel sequencing. We investigated the variability of 420 samples from a very admixed population such as Brazilians by using this approach. Considering a segment of about 7kb, 63 variable sites were detected, arranged into 75 extended haplotypes. We detected 37 different promoter sequences (but few frequent ones), 27 different coding sequences (15 representing new HLA-E alleles) and 12 haplotypes at the 3′UTR segment, two of them presenting a summed frequency of 90%. Despite the number of coding alleles, they encode mainly two different full-length molecules, known as E*01:01 and E*01:03, which corresponds to about 90% of all. In addition, differently from what has been previously observed for other non classical HLA genes, the relationship among the HLA-E promoter, coding and 3′UTR haplotypes is not straightforward because the same promoter and 3′UTR haplotypes were many times associated with different HLA-E coding haplotypes. This data reinforces the presence of only two main full-length HLA-E molecules encoded by the many HLA-E alleles detected in our population sample. In addition, this data does indicate that the distal HLA-E promoter is by far the most variable segment. Further analyses involving the binding of transcription factors and non-coding RNAs, as well as the HLA-E expression in different tissues, are necessary to evaluate whether these variable sites at regulatory segments (or even at the coding sequence) may influence the gene expression profile.

Association of HLA-G 3′UTR polymorphisms and haplotypes with severe sepsis in a Brazilian population

BackgroundThe human leukocyte antigen G (HLA-G) is a molecule involved in immune system modulation, acting in the maintenance of a state of immune tolerance. Some polymorphisms in the HLA-G gene 3′ untranslated region (3′UTR) were associated to distinct levels of HLA-G expression and to sepsis development. In the present study, haplotypes and polymorphisms of the HLA-G 3′UTR were analyzed in Brazilian septic patients. MethodsThe HLA-G 3′UTR was amplified by PCR, sequenced and eight polymorphisms were genotyped (the 14bp insertion/deletion, +3003T/C, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G and+3196C/G) in DNA samples from septic patients (with severe sepsis or septic shock) and controls. The haplotypes were inferred and association tests were performed through Chi square test and binary logistic regression. ResultsThe+3027AC genotype was associated asa risk factor to sepsis development (OR 3.17, PBonferroni 0.048). Further, the presence of the UTR-7 haplotype (OR 2.97, PBonferroni 0.018), and of 14bp-Ins_+3142G_+3187A haplotype (OR 2.39, PBonferroni 0.045) were associated with sepsis, conferring susceptibility. ConclusionOur data confirm an important role of HLA-G 3′UTR polymorphisms in the development of severe forms of sepsis (severe sepsis and septic shock). The genotyping of HLA-G genetic variants and haplotypes could be useful as a prediction tool of increased risk to severe sepsis.

P191 A first evaluation of the HLA-A gene 3′ untranslated region diversity in Brazil and its relationship with coding haplotypes

Aim Here we propose a NGS strategy to evaluate the HLA-A coding and 3′UTR variability and get reliable haplotypes. Then, we present the data obtained in very admixed population (Brazil). Methods The HLA-Agene segment was amplified in a unique amplicon considering 408 Brazilian individuals. Amplicons were sequenced by NGS and data processed using hla-mapper and a local pipeline to get genotypes and haplotypes. Allele and haplotype frequencies, together with Linkage Disequilibrium (LD) plots, were also calculated. Results According to the human genome draft version hg38, and known HLA-A transcripts available, the HLA-A 3′UTR comprises a segment of 433 nucleotides. However, only 70 percent of this segment is considered at the IPD-IMGT/HLA database. A total of 21 variable sites were found in the 3′UTR and, together, they were organized in 11 different haplotypes. Seven of those variants were detected outside the segment tracked by the IMGT/HLA database. We observed a high LD throughout the HLA-A locus and associations between each of these 3′UTR haplotypes and specific coding alleles. Thus, in many cases, it is possible to detect the HLA-A allele group by targeting one or two variations at the 3′UTR. Although few haplotypes were detected, they were quite frequent. It is possible that the variability detected at this segment would influence allelic expression, mainly by posttranscriptional mechanisms through microRNA binding. Conclusions The HLA-A locus is one of the most variable genes on the human genome. Most of its variability was described within exons 2 and 3, which encode the peptide-binding groove. Thus, these segment are usually the only ones targeted by many HLA-A studies. Little is known regarding the HLA-Aregulatory segments variability, including its 3′ untranslated region (3′UTR). Thus, HLA-A 3′UTR present few but frequent haplotypes, and each of these haplotype is associated with a specific group of coding alleles, which in turn may present a similar post transcriptional profile.

Immunomodulatory Role of HLA-G in Hematopoietic Stem Cell Transplantation Outcome

Introduction: HLA-G, the non-classical HLA class I molecule, is encoded by the human MHC complex at chromosome 6. Limited polymorphism, restricted expression and presence as isoforms are unique features of this immunomodulatory molecule that interacts with receptors ILT-2/ILT-4/KIR2DL4 on immune cells and creates a tolerogenic microenvironment. HLA-G expression and function is influenced by the polymorphism in 3’UTR region where different miRNA interact and impact mRNA stability. Objectives: The present study aimed to evaluate the clinical relevance of HLA-G in Hematopoietic Stem Cell Transplantation (HSCT). Material and Methods: We enrolled 50 North Indian patients (diagnosed with acute leukemia or aplastic anemia) who underwent HSCT and 202 healthy controls belonging to same ethnicity. We investigated HLA-G 3’UTR exon8 polymorphism, 14 bp insertion/deletion (ins/del) and SNPs. Exon8-14 bp ins/del was detected using sequence specific primers and SNPs were identified by sequencing. The soluble HLA-G levels and Treg cell frequency were estimated at pre and post transplant days 15,30,60,90,GvHD. Results: In this cohort, although the 14 bp ins/del genotype was predominant among controls, the sHLA-G levels were highest in those carrying14bp ins/ins genotype. Based on SNP linkage, 8 UTR haplotypes were identified. The sHLA-G levels were higher in individuals with 14 bp-INS linked haplotypes UTR2 and UTR4, in comparison to those with UTR 1, 6 and 7 (intermediate levels) and with 14 bp-DEL linked haplotypes UTR3 and UTR5 (low level). 40% of HSCT recipients experienced graft versus host disease (GvHD). Recipients with 14 bp ins/ins genotype had lower incidence of GvHD compared to those with del/del or ins/del genotype. Higher percentage of recipients carrying HLA-G 3’UTR SNPs +3027 CC vs CA/AA, +3035 CC vs TT/TC experienced GvHD. Majority of recipients carrying UTR1 and UTR3 developed GvHD. Further, sHLA-G levels were significantly lower at the time of GvHD, compared to the pre and post-transplant time points. The Treg cell frequency also correlated with sHLA-G levels. Discussion: GvHD is the major cause of morbidity and mortality post HSCT. Investigating HLA-G 3’UTR polymorphism, sHLA-G levels and Treg cell frequency may be beneficial for transplant monitoring. HLA-G molecule could be proposed as possible biomarker for predicting GvHD and HSCT outcome. All India Institute of Medical Sciences, New Delhi. Indian Council of Medical Research, India.

Significance of 3'UTR and Pathogenic Haplotype in Glucose-6-Phosphate Deficiency.

To determine the polymorphisms in the 3' untranslated region (3'UTR) of the G6PD gene and analyze the specific SNPs or haplotypes that may affect messenger RNA (mRNA) secondary structure by in silico analysis. We studied the 3'UTR polymorphisms in 107 healthy subjects living in Thailand (ethnicities unknown). The haplotype was analyzed using Haploview software. mRNA secondary structure and microRNA binding of the G6PD gene were predicted by use of the CLC Main Workbench 6.9 and RegRNA 2.0 programs. The results revealed 1 new variant in the 3'UTR of the G6PD gene (c.*99A > G). Haplotype ATCG showed significance by association-test results that yielded a high odds ratio (OR, 7.90; 95% confidence interval [CI], 3.15-19.81). Moreover, this haplotype affected G6PD mRNA secondary structure changes and microRNA binding via in silico analysis. These results suggest that the haplotype ATCG is associated with reduction of G6PD enzyme expression, G6PD mRNA secondary structure changes, and microRNA binding via in silico analysis.

Haplotypes of the HLA-G 3' Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially.

The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5’ and 3’ regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3’ untranslated region (3’UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3’UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3’UTR haplotypes in healthy individuals and reinforce that 3’UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G.

HLA-G coding region and 3′untranslated region (3′UTR) in two Chinese Han populations

In this study, exons 2–4 and 3′untranslated region (3′UTR) of human leukocyte antigen (HLA)-G gene were investigated for 201 and 104 healthy unrelated Han samples recruited from Hunan Province, southern China and central Inner Mongolia Autonomous Region, northern China, respectively, using sequence-based typing and cloning methods. Totally 12 HLA-G alleles in the coding region, 9 variable sites in 3′UTR, 8 3′UTR haplotypes and 15 HLA-G extended haplotypes (EHs) incorporating the coding region and 3′UTR were observed. Very strong linkage disequilibrium (LD) was observed between HLA-A and HLA-G, and between HLA-G coding region and 3′UTR in each population (all global P=0.0000). Seven HLA-A-G haplotypes showed significant LD in both populations. Three HLA-G alleles in the coding region, 4 polymorphic sites in the 3′UTR, 3 3′UTR haplotypes and 4 HLA-G EHs differed significantly in their distributions between the 2 Chinese Han populations (all P≤0.0001). There was evidence for balancing selection acting on HLA-G 3′UTR positions +3010, +3142 and +3187 in the two populations. The NJ dendrograms demonstrated the existence of two basic HLA-G lineages and indicated that, HLA-G*01:01:01, the most common HLA-G allele, formed a separate lineage from other alleles. Our results shed new lights into HLA-G genetics among Chinese Han populations. The findings reported here are of importance for future studies related to post-transcriptional regulation of HLA-G allelic expression and the potential role of HLA-G in disease association in populations of Chinese ancestry.

3’UTR SNPs and Haplotypes in the GATA4 Gene Contribute to the Genetic Risk of Congenital Heart Disease

Introduction and objectivesSingle-nucleotide polymorphisms within a microRNA binding site can have different effects on gene expression, influencing the risk of disease. This study aimed to evaluate the association between single-nucleotide polymorphisms and haplotypes in the 3’UTR of the GATA4 gene and congenital heart disease risk. MethodsBioinformatics algorithms were used to analyze single-nucleotide polymorphisms in putative microRNA-binding sites of GATA4 3’UTR and to calculate the difference in free energy of hybridization (ΔFE, kcal/mol) for each wild-type vs the variant allele. ResultsThe study population comprised 146 Caucasian patients (73 males; 6.68 ± 7.79 years) and a 265 healthy newborn participants (147 males). The sum of all |ΔFE| was considered to predict the biological importance of single-nucleotide polymorphisms binding more microRNAs. Next, the 4 polymorphisms (+1158C > T, +1256 A > T, +1355 G > A, +1521C > G) with the highest predicted |ΔFEtot| (9.91, 14.85, 11.03, 21.66kcal/mol, respectively) were genotyped in a case-control study (146 patients and 250 controls). Applying a correction for multiple testing only the +1158 T allele was found to be associated with a reduced risk showing significant difference between patients and controls. Haplotype analysis showed that the T-T-G-C haplotype (more uncommon in congenital heart diseases than in controls) was associated with a significantly decreased risk (P = .03), while the rare C-A-A-C haplotype, which was very uncommon in controls (0.3%) compared with the disease (2.4%), was associated with a 4-fold increased risk of disease (P = .04). ConclusionsCommon variants in 3’UTR of the GATA4 gene jointly interact, affecting the congenital heart disease susceptibility, probably by altering microRNA posttranscriptional regulation.