Revealing complete complex KIR haplotypes phased by long-read sequencing technology.

The killer cell immunoglobulin-like receptor (KIR) region of human chromosome 19 contains up to 16 genes for natural killer (NK) cell receptors that recognize human leukocyte antigen (HLA)/peptide complexes and other ligands. The KIR proteins fulfill functional roles in infections, pregnancy, autoimmune diseases and transplantation. However, their characterization remains a constant challenge. Not only are the genes highly homologous due to their recent evolution by tandem duplications, but the region is structurally dynamic due to frequent transposon-mediated recombination. A sequencing approach that precisely captures the complexity of KIR haplotypes for functional annotation is desirable. We present a unique approach to haplotype the KIR loci using single-molecule, real-time (SMRT) sequencing. Using this method, we have-for the first time-comprehensively sequenced and phased sixteen KIR haplotypes from eight individuals without imputation. The information revealed four novel haplotype structures, a novel gene-fusion allele, novel and confirmed insertion/deletion events, a homozygous individual, and overall diversity for the structural haplotypes and their alleles. These KIR haplotypes augment our existing knowledge by providing high-quality references, evolutionary informers, and source material for imputation. The haplotype sequences and gene annotations provide alternative loci for the KIR region in the human genome reference GrCh38.p8.

Different DRB1*03:01-DQB1*02:01 haplotypes confer different risk for celiac disease.

Celiac disease is associated with the HLA-DR3-DQA1*05:01-DQB1*02:01 and DR4-DQA1*03:01-DQB1*03:02 haplotypes. In addition, there are currently over 40 non-HLA loci associated with celiac disease. This study extends previous analyses on different HLA haplotypes in celiac disease using next generation targeted sequencing. Included were 143 patients with celiac disease and 135 non-celiac disease controls investigated at median 9.8 years (1.4-18.3 years). PCR-based amplification of HLA and sequencing with Illumina MiSeq technology were used for extended sequencing of the HLA class II haplotypes HLA-DRB1, DRB3, DRB4, DRB5, DQA1 and DQB1, respectively. Odds ratios were computed marginally for every allele and haplotype as the ratio of allelic frequency in patients and controls as ratio of exposure rates (RR), when comparing a null reference with equal exposure rates in cases and controls. Among the extended HLA haplotypes, the strongest risk haplotype for celiac disease was shown for DRB3*01:01:02 in linkage with DQA1*05:01-DQB1*02:01 (RR = 6.34; P-value < .0001). In a subpopulation analysis, DRB3*01:01:02-DQA1*05:01-DQB1*02:01 remained the most significant in patients with Scandinavian ethnicity (RR = 4.63; P < .0001) whereas DRB1*07:01:01-DRB4*01:03:01-DQA1*02:01-DQB1*02:02:01 presented the highest risk of celiac disease among non-Scandinavians (RR = 7.94; P = .011). The data also revealed 2 distinct celiac disease risk DR3-DQA1*05:01-DQB*02:01 haplotypes distinguished by either the DRB3*01:01:02 or DRB3*02:02:01 alleles, indicating that different DRB1*03:01-DQB1*02:01 haplotypes confer different risk for celiac disease. The associated risk of celiac disease for DR3-DRB3*01:01:02-DQA1*05:01-DQB1*02:01 is predominant among patients of Scandinavian ethnicity.

Establishment of optimized ELISA system specific for HLA-G in body fluids.

Recently, human leukocyte antigen-G (HLA-G) has been a focus in the field of reproductive immunology, tumor progression and transplantation, because of its inhibitory function as ligand to the inhibitory receptors leukocyte immunoglobulin-like receptors (LILR) B1 and LILRB2. The HLA-G is expressed in distinct mRNA isoforms, one of which encodes a soluble HLA-G (sHLA-G) protein, detectable by sandwich ELISA. Therefore, sHLA-G ELISAs have been used as a noninvasive diagnosis system. While a number of sHLA-G-specific ELISAs have been described, our prior studies showed that data obtained by the conventional ELISA system detecting sHLA-G in body fluids was not consistent with the data obtained from immunoprecipitation (IP)/immunoblotting (IB). Therefore, we established an optimized ELISA system described in this report, which yields results consistent with IP/IB analysis. Using this system, we determined sHLA-G protein in amniotic fluids, and found that sHLA-G levels at preterm (∼36 weeks) were clearly higher than those at term (37-41 weeks). These data and supporting experiments showed that the ELISA system we established can be an useful tools for the detection of sHLA-G protein in body fluids than the conventional ELISA system.

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.

An update to HLA nomenclature, 2010.

The WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports.

Resequencing of the human major histocompatibility complex in patients with rheumatoid arthritis and healthy controls in Alaska Natives of Southeast Alaska.

High prevalence and severity of rheumatoid arthritis (RA) with an early age of onset have previously been described in Alaska Native and American Indian (AN/AI) populations. The contribution of HLA-DRB1 alleles encoding a similar amino acid sequence, referred to as the shared epitope (SE), to RA risk is well recognized in multiple populations worldwide. DRB1*1402 allele is the major SE-encoding allele in AN/AI populations. However, DRB1*1402 is highly prevalent in healthy Alaska Natives of Southeast Alaska (AN), with no significant difference from RA patients, indicating this allele alone is not informative for defining genetic risk and non-human leukocyte antigen (non-HLA) genes are likely important in AN. We sought to deep resequence the human major histocompatibility complex (MHC) to characterize the single-nucleotide polymorphism (SNP) haplotypes within this region in RA cases and controls in AN. Approximately 99 kb of the MHC was resequenced with 95 amplicons throughout this region. Thirty-four cases and 74 controls were examined. A total of 696 SNPs were discovered from 85 of the selected 95 amplicons. Disease association signals were detected for nine of the 95 amplicons analyzed. Increased risk of RA was associated with five amplicons in the class I, class II or class III region and resistance to disease with four amplicons in the class I region. Our results indicate that non-HLA MHC genes and/or unknown exogenous factors likely modulate risk of RA in the AN population.

Definitive high resolution typing of HLA-E allelic polymorphisms: Identifying potential errors in existing allele data.

A set of robust PCR-SSP reactions were developed for each of the five polymorphic sites that define the five alleles of the HLA class Ib gene, HLA-E. This method was developed using 28 homozygous cell lines and further tested in a sample of African-Americans, a sample of Japanese, and a core panel of cell lines compiled for the 13th International Histocompatibility Workshop. Three alleles were found in each of these four sample groups, HLA-E*0101 (64.29, 50.00, 32.00 and 56.58%, respectively), *01031 (5.36, 20.65, 39.00 and 18.42%) and *01032 (30.35, 29.35, 29.00, and 25.00%). HLA-E*0102 was not detected in any of these samples nor in the cell line, LCL 722.221, in which this allele was originally described. HLA-E*0104 was not found either. This latter allele was originally reported in Japanese at a frequency of 1/22 (4.5%), which should have been high enough to have resulted in multiple occurrences of the *0104 allele in the samples tested in this study. We propose that the existence of the HLA-E*0102 and E*0104 alleles should be questioned.

Soluble HLA-G circulates in maternal blood during pregnancy.

OBJECTIVE: Soluble isoforms of the HLA class Ib gene HLA-G have been identified at the maternal-fetal interface. Because soluble forms of other HLA class I antigens modulate T-cell reactivity and induce cellactivated apoptosis, our goal was to determine whether soluble HLA-G circulates in maternal or fetal blood and to identify the specific isoform. STUDY DESIGN: Capture enzyme-linked immunosorbent assays with mouse monoclonal antibodies directed toward an epitope present on all isoforms of soluble HLA-G were constructed to identify soluble HLA-G in 44 serum samples from nonpregnant control subjects, 129 serum samples from pregnant women, and 10 samples of term cord blood. Distinguishing between soluble HLA-G1, which is composed of heavy chains complexed with light chains (beta(2)-microglobulin), and soluble HLA-G2, which consists only of heavy chains, was achieved by substituting a monoclonal antibody that requires beta(2)-microglobulin for binding (W6/32) in the capture phase of the enzyme-linked immunosorbent assay. RESULTS: Capture enzyme-linked immunosorbent assays with mouse anti-soluble HLA-G showed that soluble HLA-G was present at all stages of gestation and that levels of soluble HLA-G were statistically significantly higher in serum samples from pregnant women than in serum samples from nonpregnant women. In contrast, W6/32 failed to detect soluble HLA-G in serum samples from pregnant women. Cord serum samples did not contain detectable soluble HLA-G. CONCLUSION: Collectively, the data indicate that pregnancy is characterized by the presence of soluble HLA-G circulating in maternal blood and strongly suggest that the major isoform is soluble HLA-G2.

Melanomas and melanoma cell lines do not express HLA-G, and the expression cannot be induced by gammaIFN treatment.

HLA-G is an effective ligand of natural killer (NK) inhibitory receptors, HLA-G transcripts have been detected in several human tumors, and cytokines like gamma interferon (IFN) enable HLA-G molecules to be expressed. These findings are particularly upsetting in case of melanomas: IFN treatment is frequently included in melanoma therapeutic protocols, and downregulation of classical class I molecules occurs in nearly half of these tumors. Therefore, a melanoma cell downregulating classical class I and de novo expressing HLA-G, either constitutively or upon IFN treatment, is probably a stealthy target for the immune system, having inhibited both the cytotoxic T lymphocyte (CTL) and the NK activity. To elucidate this point we have investigated the expression of HLA-G molecules in 45 melanoma cell lines before and after gammaIFN treatment. Analysis was performed by immunofluorescence and flow cytometry, using the anti-HLA-G MoAbs 87G and G233, by Western blot, using the anti-HLA-G MEM/G1 MoAb and PAG1 antiserum, and by RT-PCR analysis. In addition, 8 melanoma tissues from patients free from therapy and 6 nevi were studied by immunohistochemistry using the 87G MoAb. No evidence was gathered of HLA-G expression, neither constitutive nor, in cell lines, after gammaIFN treatment. We therefore conclude that HLA-G expression is an uncommon event in melanomas, and that a therapy including IFNs cannot harm the patient by inducing the de novo expression of HLA-G molecules at least in its G1 isoform.

Human cytomegalovirus gene products US3 and US6 down-regulate trophoblast class I MHC molecules.

The epidemiological correlation between human CMV (HCMV) infection and spontaneous fetal loss has been suggested, but the underlying mechanism is not well understood. Fetal cytotrophoblasts, which are in direct contact with the maternal immune system in the uterus during pregnancy, do not express HLA-A and HLA-B, but express the nonclassical class I HLA-G and HLA-C. It has been shown that both HLA-G and HLA-C are capable of inhibiting NK-mediated cell lysis. In our present study, using human trophoblast cell lines as well as other cell lines stably transfected with the human class I genes, we have demonstrated that HCMV US3 and US6 down-regulate the cell-surface expression of both HLA-G and HLA-C by two different mechanisms. HCMV US3 physically associates with both trophoblast class I MHC species, retaining them in the endoplasmic reticulum. In contrast, HCMV US6 inhibits peptide transport by TAP and thus specifically the intracellular trafficking of class I molecules. Therefore, these findings suggest for the first time a possible molecular mechanism underlying HCMV-related spontaneous pregnancy loss.

HLA-G in reproduction: studies on the maternal-fetal interface.

For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation. A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera. Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2. To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems. Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes. Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4. Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy.

Data acquisition, data storage, and data presentation in a modern genetics laboratory.

Modern genetic analysis can be divided into three main areas of investigation. The first is data acquisition, in the form of genomic sequence and the cataloguing of polymorphism data of the single nucleotide polymorphism variety (so called SNPs). Once identified, such genetic information can be adapted into high throughput tests to examine genetic information in large populations, making the analysis of sufficiently large numbers both cost and time effective so that relatively low-penetrant genetic effects can be accurately detected. The third step is correlating variation with phenotype (e.g. disease susceptibility or resistance) for a variety of disorders is paramount in our motivation and indeed is a common goal of modern human genetic analysis. While the technology to acquire vast amounts of genetic data is now well established and continues to expand, the ability to deal with such data, from the process of acquisition, storage, and analysis depends fundamentally on a solid informatics infrastructure as an essential component. Indeed, most of the major gains in productivity in this field are to be realized on the informatics front, and involve automating data acquisition, defining and sorting data in databases for quality control and analysis and facilitating access to data for the large variety of data analyses. Informatics-related issues including those relating to data acquisition, database structure, and analysis tools are summarized here in an effort to define some of the issues relevant to establishing informatics infrastructure in a small genetics laboratory focused on resequencing human immune response genes. From inherited diseases to drug efficacy to the specific genetic changes occurring during tumor development, this new field of medical genetics promises a profound impact on the state of human health. Ultimately, any and all advances in this field will continue to depend on major investments in informatics.