Evaluation of HLA‐G5 Plasmatic Levels During Pregnancy and Relationship with the 14‐bp Polymorphism

Citation Gonzalez A, Alegre E, Torres MI, Díaz‐Lagares A, Lorite P, Palomeque T, Arroyo A. Evaluation of HLA‐G5 plasmatic levels during pregnancy and relationship with the 14‐bp polymorphism. Am J Reprod Immunol 2010 Problem Plasmatic HLA‐G levels increase during pregnancy, but the contribution of each different isoform has not been elucidated yet. Method of study HLA‐G5 was analyzed by ELISA in 19 controls, 79 women in the first 8 weeks of pregnancy and in nine women monthly until delivery. Genotyping for the 14‐bp polymorphism was performed by PCR amplification of exon 8. Results HLA‐G5 was detected in plasma from 80% of pregnant women. The levels did not change during pregnancy, and there were no differences compared to control non‐pregnant women. There was a high interindividual variation that was maintained throughout the pregnancy. The presence of +14‐bp allele was associated with HLA‐G5 positivity. Pregnant women who were heterozygotic to 14‐bp polymorphism had significantly higher levels of HLA‐G5 compared to ?14 bp/?14‐bp homozygotic. Conclusion Plasmatic HLA‐G5 levels do not change during pregnancy and its concentration depends on 14‐bp polymorphism.     

Urotensin-II 143 G/A polymorphism is not associated with the risk of preeclampsia in Korean women

Citation Na S, Shim J‐Yoon, Jung B‐Kyung, Won H‐Sung, Lee PRyang, Kim A. Urotensin‐II 143 G/A polymorphism is not associated with the risk of preeclampsia in Korean women. Am J Reprod Immunol 2011; 66: 423–427 Problem Urotensin‐II (UTS‐II) may be associated with preeclampsia. We therefore assessed whether the UTS‐II 143 G/A polymorphism is associated with preeclampsia in Korean women. Method of study This prospective case–control study enrolled 109 patients with preeclampsia and 144 healthy pregnant controls, all of whom were genotyped for the UTS‐II 143 G/A polymorphism using the real‐time TaqMan SNP Genotyping Assay. Results There was no significant difference in the distribution of the 143 G/A polymorphism or the frequency of the 143 A allele between women with preeclampsia and controls. In a dominant model, carriers of the A allele were not significantly more prevalent in the preeclamptic (53.2%) than in the control (60.4%) group. In addition, subgroup analysis showed no significant difference in genotype distribution or allelic frequency of the 143 G/A polymorphism between women with mild or severe preeclampsia and controls. Conclusion In Korean women, the common UTS‐II 143 G/A polymorphism is unlikely to have an association with the risk of preeclampsia.     

HLA‐G gene 14‐bp deletion variant protects Iranian subjects against chronic hepatitis B infection

To investigate whether 14‐bp Ins/Del polymorphism in HLA‐G gene is associated with the risk of chronic hepatitis B (CHB) infection. This study was performed on a total of 396 individuals including 199 CHB patients and 197 healthy subjects from a south‐east Iranian population. We genotyped 14‐bp Ins/Del polymorphism in the HLA‐G gene using polymerase chain reaction method. The results of our study revealed that the HLA‐G 14‐bp deletion polymorphism was associated with a reduced risk of CHB at both allele and genotypic levels. The 14‐bp Del allele and Ins/Del genotype were more frequent in control group than in CHB patients (37% vs 28% for Del allele with OR = 0.68 and p‐value = .015; 73% vs 52% for Ins/Del genotype with OR = 0.43 and p‐value = .001) and both were protective factors against CHB. However, no difference was found in the distribution of HLA‐G 14‐bp genotypes among subjects with varied levels of HBV DNA or hepatic enzymes (p > .05). Our findings, for the first time, suggest that the HLA‐G 14‐bp Ins/Del polymorphism may be a marker for genetic susceptibility to CHB infection.     

The 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene is associated with severe preeclampsia

Preeclampsia is associated with thrombosis of the intervillous or spiral artery. A deletion/insertion polymorphism (4G or 5G) in the promoter of the plasminogen activator inhibitor type 1 (PAI-1) gene is suggested to be involved in regulating the synthesis of the inhibitor, 4G allele, being associated with the enhanced gene expression and plasma PAI-1 levels. We assessed the association between preeclampsia and the 4G/5G polymorphism of the PAI-1 gene in 115 preeclamptic patients, 210 pregnant controls, and 298 healthy volunteer controls. The frequency of the homozygotes for the 4G allele was significantly higher in the patients than in the control pregnant women (P = 0.04) or in the healthy volunteers (P = 0.02). The 4G allele frequency was also significantly higher in the patients than in the control group of pregnant women (P = 0.03) and in the healthy volunteers (P = 0.02). These results suggest that the presence of the 4G/4G genotype of the PAI-1 gene is one of the risk factors for preeclampsia. Received: October 14, 1999 / Accepted: January 4, 2000     

Human leucocyte antigen‐G 14‐bp InDel polymorphism and oral squamous cell carcinoma risk in Chinese Han population: A case–control study

Human leucocyte antigen‐G (HLA‐G) is a nonclassical HLA class I molecule involved in tumour immune escape. The purpose of this study was to investigate the association between the 14‐bp insertion/deletion (InDel) polymorphism in the 3′ untranslated region (3′‐UTR) of HLA‐G gene and oral squamous cell carcinoma (OSCC) risk in Chinese Han population (216 cases and 193 healthy controls), and furthermore, to evaluate serum soluble HLA‐G (sHLA‐G) levels in the OSCC patients. Our results demonstrated that the Ins allele was significantly less frequent in the OSCC patients than that in the healthy controls (odds ratio [OR] = 0.75; 95% confidence interval [CI]: 0.57–0.99; p = 0.040). Distribution of the 14‐bp genotypes in the OSCC patients and the healthy controls revealed that the Ins/Ins genotype was associated with decreased OSCC risk in both the codominant model (Ins/Ins versus Del/Del; OR = 0.57; 95% CI = 0.33–0.99; p = 0.044) and the log‐additive model (OR = 0.76; 95% CI: 0.58–0.99; p = 0.044). The serum sHLA‐G level was significantly higher in the OSCC patients than those in the healthy controls (p < 0.001). Receiver operating characteristic (ROC) curve revealed the valuable diagnostic value of sHLA‐G for OSCC detection, with an area under the ROC curve (AUC) of 0.891 (95% CI: 0.856–0.925, p < 0.001). The OSCC patients with Ins/Ins genotype had lower serum sHLA‐G levels than those with Ins/Del and Del/Del genotypes (p = 0.015). Furthermore, serum sHLA‐G levels were significantly increased with the increasing TNM stages of the OSCC patients (p = 0.017). Our findings revealed that the HLA‐G 14‐bp InDel polymorphism might be a genetic risk factor for OSCC susceptibility, and the serum sHLA‐G may act as a promising biomarker for noninvasive diagnosis of OSCC.     

Calcium‐sensing receptor gene polymorphism (rs7652589) is associated with calcium nephrolithiasis in the population of Yi nationality in Southwestern China

The calcium‐sensing receptor (CaSR) gene plays an important role in regulating the Ca²⁺ balance and reducing the risk for calcium stones. In this study, we evaluated the association of CaSR polymorphisms with calcium nephrolithiasis in the population of Yi nationality in Southwestern China. Biochemical variables were evaluated in 624 calcium nephrolithiasis patients and 470 age‐matched healthy controls without a history of nephrolithiasis. CaSR polymorphisms rs7652589, rs1501899, rs1801725 (Ala986Ser), rs1042636 (Arg990Gly) and rs1801726 (Gln1011Glu) were investigated between the calcium nephrolithiasis patients and healthy controls, using direct sequencing. Compared with the healthy controls, serum creatinine and 24‐hour urine calcium levels were significantly higher in calcium nephrolithiasis patients. Among these five polymorphisms, the genotypic and allelic frequency distributions of rs7652589 SNP was significantly associated with the risk of calcium nephrolithiasis. However, there were no genotypic or allelic distribution differences for rs1501899, rs1801725, rs1042636, and rs1801726 polymorphisms between calcium nephrolithiasis patients and healthy controls. Moreover, the association between rs7652589 SNP genotypes and the biochemical variables was not found. Our study showed that CaSR rs7652589 polymorphism had a significant effect on the risk of developing calcium nephrolithiasis in the population of Yi nationality in Southwestern China.     

Distribution of HLA‐G 14‐bp insertion/deletion polymorphism in six Chinese ethnic groups

Recently, a 14‐bp insertion/deletion polymorphism (+14 bp/?14 bp) in exon 8 of the Human leucocyte antigen‐G (HLA‐G) gene has been studied extensively because this polymorphism has been associated with HLA‐G mRNA stability and could influence HLA‐G mRNA expression. We investigated the distribution of the 14‐bp insertion/deletion polymorphism in six different Chinese ethnic groups (Bulang, Wa, Hani, Jinuo, Maonan and Zhuang), which originated from three major ancient tribes (Di‐Qiang, Baipu and Baiyue) in China. Comparison of the 14‐bp insertion frequency in the six groups with other Chinese groups showed marked variation among the three ancient tribes, Di‐Qing (0.490–0.534), Baipu (0.470–0.609) and Baiyue (0.280–0.344). Furthermore, the frequencies of the 14‐bp insertion were similar in groups that came from the same ancient tribe, which indicated that the individuals who share the 14‐bp insertion have the most probably inherited the 14‐bp element from a common ancestor. In addition, an intra‐tribal comparison of the 14‐bp insertion/deletion frequencies between the descendants of the ancient ancestral tribes suggests that population histories or some environmental effects, such as founder effect or isolation, might also influence the distribution.     

HLA‐G regulatory variants and haplotypes with susceptibility to recurrent pregnancy loss

HLA‐G is a nonclassical Class I major histocompatibility complex (MHC) gene. This gene has a limited protein alteration that is produced by alternative splicing and can be important in the preservation of pregnancy. Recent findings suggest that alteration in HLA‐G gene expression can lead to pregnancy failure, such as recurrent pregnancy loss (RPL). As the promoter SNPs of the gene may impact the HLA‐G expression levels, the study of these SNPs is very important. In this study, for the promoter region of HLA‐G gene in the case group (100 women with a history of two or more repeated miscarriages) and the control group (100 women with at least two successful pregnancies), PCR reaction was performed. Thereafter, PCR products were sequenced and the results were compared between the two groups. The results showed that ?1573T>C and ?1746C>A SNPs in the promoter of the HLA‐G gene associated with RPL. The outcome of the haplotype analysis also showed that the association of two haplotypes, including H1 (ATCCAGGTACGCAA) and H2 (CTTCGAGAACGCAG) with RPL, is significant. The results showed that H1 is associated with a decreased and H2 is associated with an increased risk of RPL. These results indicate the importance of the HLA‐G promoter SNPs in the pregnancy outcome. But to reach a more definite conclusion, subsequent studies on 3′ UTR and other positions with polymorphism in the 5′ UTR regions larger samples are necessary.     

Co‐expression of HLA‐B7 and HLA‐B27 alleles is associated with B7‐restricted immunodominant responses following influenza infection

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2‐K^?/?/D^?/? double‐knockout transgenic mice expressing either one or two human MHC‐I alleles. We hypothesized that co‐expression of different allele combinations figures critically in immunodominance and examined this in influenza‐infected, double Tg MHC‐I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2‐restricted matrix I.58–66, the B7‐restricted NP418–426 or the B27‐restricted NP383–391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu‐infected B7/B27 mice, a significantly reduced level of B27/NP383‐restricted CTL response was detected while there was no change in the B7/NP418‐restricted CTL response. Flu‐specific tetramer studies revealed a partial deletion of Vβ8.1⁺ NP383/B27‐restricted CD8⁺ T cells, and a diminished Vβ12⁺ CD8⁺ T‐cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co‐dominant expression of MHC‐I alleles for host immune response to pathogens.     

HLA‐G 14‐bp polymorphism affects the age of onset in Type I Diabetes Mellitus

Type I diabetes mellitus (T1DM) is an organ‐specific autoimmune disorder affecting the insulin‐producing pancreatic cells. T1DM genetic association studies have so far revealed the involvement of more than 40 loci, with particularly strong associations for the human leucocyte antigens (HLA). Further to the well‐established HLA class II associations, the immunomodulatory elements in the telomeric major histocompatibility complex locus, specifically nonclassical HLA class I, were also associated with T1DM, either in conferring susceptibility or by contributing to the overall pathogenesis. This study investigates the involvement of a 14‐bp deletion polymorphism (rs371194629) at the 3′ untranslated region of HLA‐G in the context of T1DM and age of onset. The frequency of the polymorphism was determined in unrelated T1DM Cypriot patients and findings that emerge from this study show a strong association between the HLA‐G 14‐bp polymorphism and T1DM with respect to the age of onset. Specifically, the deletion/deletion (DEL/DEL) genotype was found to be associated with an early age of onset (P = 0.001), while the presence of the insertion allele (INS) was associated to a later age of onset (P = 0.0001), portraying a possible dominant effect over the deletion allele, a role in delaying disease onset and an overall involvement of HLA‐G in the pathogenesis of type I diabetes mellitus.     

In vivo identification of an HLA‐G complex as ubiquitinated protein circulating in exosomes

The nonclassical human leukocyte antigen‐G (HLA‐G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA‐G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA‐G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA‐G by ELISA. Complexed HLA‐G was detected in exosomes, which indicates an intracellular origin of these forms. 2D‐PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA‐G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA‐G complexes. Immunoblot analysis of exudates and exosomes with an anti‐ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA‐G. HLA‐G ubiquitination could be reproduced in vitro in HLA‐G1‐transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA‐G forms in vivo that open a new perspective in the study of HLA‐G function and analysis.     

Serum antibodies to human leucocyte antigen (HLA)‐E,HLA‐F and HLA‐G in patients with systemic lupus erythematosus (SLE) during disease flares: Clinical relevance of HLA‐F autoantibodies

T lymphocyte hyperactivity and progressive inflammation in systemic lupus erythematosus (SLE) patients results in over‐expression of human leucocyte antigen (HLA)‐Ib on the surface of lymphocytes. These are shed into the circulation upon inflammation, and may augment production of antibodies promoting pathogenicity of the disease. The objective was to evaluate the association of HLA‐Ib (HLA‐E, HLA‐F and HLA‐G) antibodies to the disease activity of SLE. The immunoglobulin (Ig)G/IgM reactivity to HLA‐Ib and β2m in the sera of 69 German, 29 Mexican female SLE patients and 17 German female controls was measured by multiplex Luminex^®‐based flow cytometry. The values were expressed as mean florescence intensity (MFI). Only the German SLE cohort was analysed in relation to the clinical disease activity. In the controls, anti‐HLA‐G IgG predominated over other HLA‐Ib antibodies, whereas SLE patients had a preponderance of anti‐HLA‐F IgG over the other HLA‐Ib antibodies. The disease activity index, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)‐2000, was reflected only in the levels of anti‐HLA‐F IgG. Anti‐HLA‐F IgG with MFI level of 500–1999 was associated with active SLE, whereas inactive SLE revealed higher MFI (>2000). When anti‐HLA‐F IgG were cross‐reactive with other HLA‐Ib alleles, their reactivity was reflected in the levels of anti‐HLA‐E and ‐G IgG. The prevalence of HLA‐F‐monospecific antibodies in SLE patients was also associated with the clinical disease activity. Anti‐HLA‐F IgG is possibly involved in the clearance of HLA‐F shed from lymphocytes and inflamed tissues to lessen the disease's severity, and thus emerges as a beneficial immune biomarker. Therefore, anti‐HLA‐Ib IgG should be considered as a biomarker in standard SLE diagnostics.