Next-generation sequencing of HLA-G based on long-range polymerase chain reaction.

Clarifying the functional roles of HLA-G and the variation in the HLA-G gene that affects the expression are increasingly important in reproduction, cancer, organ transplantation, and autoimmune diseases. The homology between HLA genes and the genetic variability within each gene complicates the design of HLA gene-specific genotyping assays. We have designed a high-throughput, cost-efficient, robust, and specific assay for sequencing the full HLA-G gene including the 5'-upstream regulatory region, introns, and the 3'-untranslated region, using the next-generation sequencing (NGS) platform Ion Torrent PGM (Thermo Fisher Scientific, Waltham, Massachusetts). Conventional sequencing methods require the design of multiple primer pairs in order to cover the entire HLA-G gene. Designing multiple primer pairs specific for the HLA-G gene that also target all known alleles is difficult. Here, we present a setup that by the use of long-range polymerase chain reaction amplifies the whole HLA-G gene in a single reaction, which only requires a single HLA-G-specific primer pair. Enzymatic DNA shearing is used to break the long-range PCR product into shorter fragments ranging from 75 to 200 bp in length that are sequenced by NGS.

Distribution of HLA-G extended haplotypes and one HLA-E polymorphism in a large-scale study of mother-child dyads with and without severe preeclampsia and eclampsia.

The etiological pathways and pathogenesis of preeclampsia have rendered difficult to disentangle. Accumulating evidence points toward a maladapted maternal immune system, which may involve aberrant placental expression of immunomodulatory human leukocyte antigen (HLA) class Ib molecules during pregnancy. Several studies have shown aberrant or reduced expression of HLA-G in the placenta and in maternal blood in cases of preeclampsia compared with controls. Unlike classical HLA class Ia loci, the nonclassical HLA-G has limited polymorphic variants. Most nucleotide variations are clustered in the 5'-upstream regulatory region (5'URR) and 3'-untranslated regulatory region (3'UTR) of HLA-G and reflect a stringent expressional control. Based on genotyping and full gene sequencing of HLA-G in a large number of cases and controls (n > 900), the present study, which to our knowledge is the largest and most comprehensive performed, investigated the association between the HLA-G 14-bp ins/del (rs66554220) and HLA-E polymorphisms in mother and newborn dyads from pregnancies complicated by severe preeclampsia/eclampsia and from uncomplicated pregnancies. Furthermore, results from extended HLA-G haplotyping in the newborns are presented in order to assess whether a combined contribution of nucleotide variations spanning the 5'URR, coding region, and 3'UTR of HLA-G describes the genetic association with severe preeclampsia more closely. In contrast to earlier findings, the HLA-G 14-bp ins/del polymorphism was not associated with severe preeclampsia. Furthermore, the polymorphism (rs1264457) defining the two nonsynonymous HLA-E alleles, HLA-E*01:01:xx:xx and HLA-E*01:03:xx:xx, were not associated with severe preeclampsia. Finally, no specific HLA-G haplotypes were significantly associated with increased risk of developing severe preeclampsia/eclampsia.

Maternal and fetal human leukocyte antigen class Ia and II alleles in severe preeclampsia and eclampsia.

A line of investigations indicate that genes in the human leukocyte antigen (HLA) complex are involved in a successful acceptance of the semiallogeneic fetus during pregnancy. In this study, associations between specific HLA class Ia (HLA-A and -B) and class II (HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1) alleles and the risk of developing severe preeclampsia/eclampsia were investigated in a detailed and large-scale study. In total, 259 women diagnosed with severe preeclampsia or eclampsia and 260 matched control women with no preeclampsia, together with their neonates, were included in the study. HLA genotyping for mothers and neonates was performed using next-generation sequencing. The HLA-DPB1*04:01:01G allele was significantly more frequent (Pc=0.044) among women diagnosed with severe preeclampsia/eclampsia compared with controls, and the DQA1*01:02:01G allele frequency was significantly lower (Pc=0.042) among newborns born by women with severe preeclampsia/eclampsia compared with controls. In mothers with severe preeclampsia/eclampsia, homozygosity was significantly more common compared with controls at the HLA-DPB1 locus (Pc=0.0028). Although the current large study shows some positive results, more studies, also with a functional focus, are needed to further clarify a possible role of the classical HLA genes in preeclampsia.

Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells.

The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complications, partly explained by HLA-G polymorphisms which are associated with differences in the alternative splicing pattern and of the stability of HLA-G mRNA. Of special importance is a 14 bp insertion/deletion polymorphism located in the 3'-untranslated region of the HLA-G gene. In the current study, we present novel evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, using a very accurate and sensitive Digital droplet PCR technique. Allelic imbalance in heterozygous samples was observed as differential expression levels of 14 bp insertion/deletion allele-specific mRNA transcripts, which was further associated with low levels of HLA-G surface expression on primary trophoblast cells. Full gene sequencing of HLA-G allowed us to study correlations between HLA-G extended haplotypes and single-nucleotide polymorphisms and HLA-G surface expression. We found that a 1:1 expression (allelic balance) of the 14 bp insertion/deletion mRNA alleles was associated with high surface expression of HLA-G and with a specific HLA-G extended haplotype. The 14 bp del/del genotype was associated with a significantly lower abundance of the G1 mRNA isoform, and a higher abundance of the G3 mRNA isoform. Overall, the present study provides original evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, which influences HLA-G surface expression on primary trophoblast cells, considered to be important in the pathogenesis of pre-eclampsia and other pregnancy complications.

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with ß2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use.

A fast and easy real-time PCR genotyping method for the HLA-G 14-bp insertion/deletion polymorphism in the 3' untranslated region.

Human leukocyte antigen-G (HLA-G) is a non-classical HLA class Ib molecule shown to exhibit immunomodulatory function in a wide range of immune-based disorders. A number of functional HLA-G gene polymorphisms have been identified, including a 14-bp insertion/deletion polymorphism in exon 8 of the 3' untranslated region of the HLA-G gene, which has been associated with HLA-G mRNA stability. Moreover, studies show that homozygosity for the 14-bp insertion/deletion polymorphism is associated with lower HLA-G mRNA and protein levels and unique alternative splicing patterns. Here, we introduce a quick and reliable method to screen for the HLA-G 14-bp insertion/deletion polymorphism using an optimized real-time polymerase chain reaction protocol. The genotyping assay has been validated by comparison with conventional methods. As results can be obtained within a few hours, the assay will have a potential for clinical use.

The 3'-untranslated region of the HLA-G gene in relation to pre-eclampsia: revisited.

Abnormal human leukocyte antigen G (HLA-G) expression may be involved in pre-eclampsia. A 14 bp insertion/deletion polymorphism exists in exon 8 of the HLA-G gene. Fetal +14/+14 bp HLA-G genotype may predispose to pre-eclampsia in the mother. Other polymorphisms, besides the 14 bp polymorphism (rs66554220), in the 3'-untranslated region (3'-UTR) (exon 8) of the HLA-G gene might be associated with severe pre-eclampsia, especially in primiparas. By haplotype-specific polymerase chain reaction amplification and DNA sequence analysis in the offspring from 50 pre-eclamptic cases and 85 controls (35 and 58 primiparas), 4 single nucleotide polymorphisms (SNPs) were detected in exon 8 of the HLA-G gene [SNP2995 (rs1710), SNP3127 (rs1063320), SNP3172 (rs9380142), and SNP3181 (rs1610696)]. Complete linkage disequilibrium between the +14 bp allele and three of the SNPs (SNP2995, SNP3127, and SNP3172) were observed. Two of the polymorphisms (SNP3172 and SNP3181) were located right before and after an AUUUA-pentamer sequence; AU-rich sequences seem to be involved in mRNA stability. However, only the genotypes of the earlier showed 14 bp polymorphism and the SNP3127 (with a C to G substitution; P = 0.008, P(C) = 0.04) were significantly associated with severe pre-eclampsia in primiparas. In conclusion, this study indicates that the +14 bp HLA-G allele defines a nearly unique exon 8 haplotype, and fetuses homozygous for this haplotype [SNP 2995(C)/SNP 3127(G)/SNP 3172(A)/SNP 3181(G)/+14 bp] are associated with severe pre-eclampsia in primiparas.

HLA-G genotype and HLA-G expression in systemic lupus erythematosus: HLA-G as a putative susceptibility gene in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease mainly mediated by the deposit of immune complexes and defects in T lymphocytes and antigen-presenting cells along with a high production of T-helper 2 cytokines. A tolerance-inducible function of nonclassical class Ib human leukocyte antigen (HLA)-G molecule in innate and adaptive cellular responses has been reported, suggesting a role in inflammatory diseases. A 14 bp sequence insertion/deletion polymorphism (rs16375) in the 3'-untranslated region of the HLA-G gene has been associated to the stability of HLA-G messenger RNA. The insertion of the 14 bp sequence seems to be associated with lower levels of soluble HLA-G (sHLA-G). The aim of this study was to evaluate the possible association of the presence of the 14 bp sequence (+14 bp) with SLE. We have HLA-G genotyped 200 SLE patients and 451 healthy control subjects (HS; Italian) and analyzed the plasma levels of sHLA-G and interleukin-10 (IL-10) in a subset of SLE patients and healthy subjects (Italian and Danish). A significant increase of the +14 bp HLA-G allele was detected in the Italian SLE patients compared with HS [P = 0.003, OR 1.44 (95% CI 1.13-1.82)]. A significant increased frequency of HLA-G +14/+14 bp and a decreased frequency of HLA-G -14/-14 bp were observed in SLE patients. There median concentration of sHLA-G was significantly lower in the plasma of SLE patients compared with that in the plasma of healthy controls (P < 0.0001). Furthermore, the results confirmed higher concentrations of IL-10-positive plasma in SLE patients. These results support a potential role for HLA-G in the susceptibility of SLE.

Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells.

OBJECTIVE: Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. DESIGN: Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from the cultures and analysed on microarrays. SETTING: A laboratory investigation using placentas obtained from a hospital delivery ward. SAMPLE: Placentas from nine healthy women were obtained. STBM vesicles were isolated from the placentas and umbilical vein endothelial cell cultures were established from the umbilical cords. METHODS: Gene expression was screened by Affymetrix GeneChips and confirmed with real-time polymerase chain reaction or enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Fold changes in gene expression levels between treated and control cultures were calculated from the microarray results. RESULTS: Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10,000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation of endothelial cells by exposure to STBM. The unfolded protein response in particular may be involved. CONCLUSIONS: STBM may influence endothelial cell function during pregnancy but STBM alone cannot account for the entire range of endothelial dysfunctions observed in pre-eclampsia.

Association between human leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy outcome.

To determine if a 14-bp deletion/insertion polymorphism in the 3'-untranslated region of exon 8 of the gene encoding human leukocyte antigen (HLA)-G in a homozygous form is associated with repeated, unsuccessful in vitro fertilization (IVF) treatments, and with increased risk of recurrent spontaneous abortions (RSA), 29 white women undergoing IVF treatments, 61 RSA women and 93 fertile controls were HLA-G genotype. The HLA-G genotype, homozygous for the presence of the 14 bp sequence in exon 8, was significantly associated with reduced fertility with respect to unsuccessful IVF treatments and increased risk of recurrent miscarriage (combined P < 0.01). The 14-bp insertion/deletion polymorphism is associated with differences in HLA-G mRNA alternative splicing and levels of HLA-G. This might affect a possible immunomodulatory role of HLA-G expression in both the mother and foetus during implantation and pregnancy.