16th IHIW: international histocompatibility working group in hematopoietic cell transplantation.

The International Histocompatibility Working Group is a collaborative international effort to understand the HLA and non-HLA genetics of the transplantation barrier. The Working Group is comprised of experts in the fields of histocompatibility and immunogenetics, hematopoietic cell transplantation and outcomes research. Data for 25 855 unrelated donor transplants were submitted in support of research studies for the 16th International Histocompatibility Workshop. Active investigation is in progress in seven key areas: the impact of HLA matching, role of race and ethnicity, identification of permissible HLA mismatches, haplotype-associated determinants, minor histocompatibility antigens, immune response genes and KIR genetics. New hypotheses for the 16th workshop were developed for immunogenetic studies in cord blood and haploidentical-related donor transplantation.

Identification of the MICA*070 allele by sequencing and phasing.

A novel MICA allele, MICA*070, was defined by sequencing. The new allele differs from the MICA*008:04 sequence in exon 2, encoding a C instead of G corresponding to cDNA nucleotide position 183. This nucleotide substitution is predicted to encode serine instead of arginine at residue 38 of the α1 domain of the MICA molecule.

Genetic variation in CXCL12 and risk of cervical carcinoma: a population-based case-control study.

CXCL12 provides a chemotactic signal-directing leucocyte migration and regulates metastatic behaviour of tumour cells. We conducted a population-based case-control study to test the hypothesis that common genetic variation in CXCL12 individual single nucleotide polymorphism (SNP) alleles and haplotypes] is associated with the risk of cervical carcinoma. Cases (n = 917) were residents of western Washington State diagnosed with invasive squamous cell cervical carcinoma (SCC), invasive adenocarcinoma or adenosquamous carcinoma, or adenocarcinoma in situ of the cervix. Control participants (n = 849) were identified from the source population by random digit telephone dialling and frequency matched to cases on county and age. Nine CXCL12 tagSNPs chosen from the SeattleSNPs database were genotyped. The minor allele of intronic SNP rs266085 was inversely associated with cervical cancer under a recessive genetic effects model (OR = 0.74, 95% CI: 0.56-0.98). Among the ten common haplotypes inferred from the nine tagSNPs, one haplotype defined by minor alleles at 5'-flanking SNP rs17885289 and rs266085, and common alleles at the other seven SNPs occurred among 7.8% of cases and 10.6% of controls (dominant model OR = 0.72, 95% CI: 0.56-0.93; recessive model OR = 0.35, 95% CI: 0.12-0.97; and log-additive model OR = 0.72, 95% CI: 0.57-0.90). A stepwise procedure identified rs17885289, rs266085 and 3'-untranslated region (UTR) SNP rs266093 as the most parsimonious subset of SNPs necessary to define the haplotype inversely associated with cervical cancer risk in our study. A 3'-UTR SNP, rs1801157, previously found to be related to HIV pathogenesis, was not associated with cervical cancer risk. Further population-based studies are warranted to confirm these associations between genetic variation in CXCL12 and cervical cancer risk.

Clinical significance of donor-recipient HLA matching on survival after myeloablative hematopoietic cell transplantation from unrelated donors.

The application of unrelated donor hematopoietic cell transplantation can be expanded with the use of mismatched donors if human leukocyte antigen (HLA) disparity does not lead to increased morbidity and mortality. The rules that govern permissibility of HLA mismatches are not well defined. The International Histocompatibility Working Group in hematopoietic cell transplantation measured the risks associated with locus-specific disparity in 4796 patients transplanted for low, intermediate, or high-risk hematologic diseases. The permissibility of a given HLA mismatch is in part defined by the locus and by disease risk.

Effect of HLA-A2 allele disparity on clinical outcome in hematopoietic cell transplantation from unrelated donors.

Population-based differences in clinical outcome after unrelated donor hematopoietic cell transplantation suggest that the significance of human leukocyte antigen (HLA) mismatching may be related to locus-specific and allele-specific differences that distinguish ethnically diverse transplant donors and recipients. We studied the risks associated with HLA-A locus mismatching in two large transplant populations from the International Histocompatibility Working Group in hematopoietic cell transplantation data set to better understand permissible and nonpermissible HLA-A mismatches.

Clinical importance of HLA-DPB1 in haematopoietic cell transplantation.

There is increasing evidence for a significant effect of human leukocyte antigen (HLA)-DPB1 mismatching on complications following unrelated donor haematopoietic cell transplantation (HCT). In this analysis of 5930 patient/donor pairs, we found that a DPB1 mismatch predicted significantly for an increased risk of acute graft-vs-host disease [hazard ratio (HR): 1.33; P-value = <0.0001], while protecting against disease relapse (HR: 0.82, P-value = 0.01). These data support an immunogenic role for HLA-DPB1 in HCT and the need for pretransplant tissue typing at this locus.

Hematopoietic stem cell transplantation: killer immunoglobulin-like receptor component.

Recognition of recipient human leukocyte antigen (HLA) class I ligand by donor natural killer cell killer immunoglobulin-like receptors (KIR) has been proposed as the basis for donor allograft reactivity against malignancy leading to reduction in posttransplant relapse and higher survival for acute myelogenous leukemia. Analysis of KIR ligand effects in 1770 patients undergoing myeloablative T-replete hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors showed that lack of KIR ligand in patients for inhibitory KIR was associated with lower hazards of relapse in leukemia patients with in HLA-mismatched transplants [hazard ratio (HR): 0.061; 95% confidence interval (CI): 0.43-0.85; P-value = 0.004]. Absence of HLA-C group 2 or HLA-Bw4 KIR ligands were each associated with lower hazards of relapse (HR: 0.47; 95% CI: 0.28-0.79; P-value = 0.004; HR: 0.56; 95% CI: 0.33-0.97; P-value = 0.04, respectively). Based on these analyses, recipient homozygosity for HLA-B or -C epitopes that define KIR ligands is likely to be a predictive factor for leukemia relapse following myeloablative HCT from unrelated donors. KIR genotyping for unrelated donors and recipients will clarify the role of these receptors in transplant outcome.

MHC class I, II, and III microsatellite marker matching and survival in unrelated donor hematopoietic cell transplantation.

Microsatellites (Msats) are effective markers for disease association mapping. The International Histocompatibility Working Group in hematopoietic cell transplantation applied Msats to determine whether potential new transplantation determinants are encoded within the major histocompatibility complex. Retrospective analysis of human leukocyte antigen-identical unrelated donor transplants provided a homogeneous population to measure Msat-associated risks of mortality.

Immune response gene polymorphisms in unrelated donor hematopoietic cell transplantation.

Immune response genes (IRG) play an important role in inflammation and control of infection after allogeneic transplantation. The International Histocompatibility Working Group (IHWG) in hematopoietic cell transplantation took a candidate gene approach to define the risks associated with genetic variation for a panel of well-characterized IRG.

MHC microsatellite diversity and linkage disequilibrium among common HLA-A, HLA-B, DRB1 haplotypes: implications for unrelated donor hematopoietic transplantation and disease association studies.

Twenty-two human major histocompatibility complex (MHC) region microsatellite (Msat) markers were studied for diversity and linkage disequilibrium (LD) with HLA loci in hematopoietic cell transplant recipients and their HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 allele-matched unrelated donors. These Msats showed highly significant LD over much of the MHC region. The Msat diversity of five common Caucasian haplotypes (HLA-A1-B8-DR3, A3-B7-DR15, A2-B44-DR4, A29-B44-DR7, and A2-B7-DR15) was examined using a new measure called 'haplotype specific heterozygosity' (HSH). Each of the five haplotypes had at least one Msat marker with an HSH value of zero indicating that only one Msat allele was observed for the particular HLA haplotype. In addition, the ability of Msats to predict HLA-A-B-DRB1 haplotypes was studied. Over 90% prediction probability of two common haplotypes (HLA-A1-B8-DR3 and HLA-A3-B7-DR15) was achieved with information from three Msats (D6S265/D6S2787/D6S2894 and D6S510/D6S2810/D6S2876, respectively). We demonstrate how the HSH index can be used in the selection of informative Msats for transplantation and disease association studies. Markers with low HSH values can be used to predict specific HLA haplotypes or multilocus genotypes to supplement the screening of HLA-matched donors for transplantation. Markers with high HSH values will be most informative in studies investigating MHC region disease-susceptibility genes where HLA haplotypic effects are known to exist.

Identification of a novel HLA-B*07 allele--HLA-B*0726.

We describe here, the identification of a novel HLA-B*07 allele named HLA-B*0726. This allele was found in a Caucasian individual serologically typed as HLA-B7, B35. Novel DNA probe patterns for the HLA-B*07 allele were found using HLA-B specific reverse sequence-specific oligonucleotide probe (SSOP) and sequence-specific primer (SSP) typing. DNA sequencing demonstrated the presence of a new HLA-B*07 sequence variant encoding a single nucleotide substitution from a G to a T at nucleotide 539 in exon 3. This results in an amino acid substitution from arginine to leucine at residue 156 in exon 3.

Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation.

BACKGROUND: Successful engraftment of hematopoietic stem cells from unrelated donors is influenced by disparities between the donor and recipient for HLA-A, B, and C alleles. Disparities between HLA sequence polymorphisms that are serologically detectable are termed antigen mismatches, whereas those that can be identified only by DNA-based typing methods are termed allele mismatches. Whether both kinds of polymorphisms are important in transplantation is not known. We tested the hypothesis that allele mismatches that are detectable only at the DNA level are less immunogenic than those that are serologically detectable and thereby are associated with a lower risk of graft failure after hematopoietic-cell transplantation METHODS: We used DNA sequencing to define the HLA-A, B, and C alleles in 471 patients who received bone marrow from unrelated donors for the treatment of chronic myeloid leukemia after myeloablative conditioning therapy. The odds ratios for graft failure were determined for recipients of transplants from donors with a single class I allele mismatch, a single class I antigen mismatch, or two or more class I mismatches, as compared with those with no mismatch RESULTS: A single HLA allele mismatch did not increase the risk of graft failure, whereas a single antigen mismatch significantly increased the risk. The risk was also increased if the recipient was HLA homozygous at the mismatched class I locus or if the donor had two or more class I mismatches CONCLUSIONS: HLA class I antigen mismatches that are serologically detectable confer an enhanced risk of graft failure after hematopoietic-cell transplantation. Transplants from donors with a single class I allele mismatch that is not serologically detectable may be used without an increased risk of graft failure.

Genomics of unrelated-donor hematopoietic cell transplantation.

Unrelated-donor hematopoietic cell transplantation is a proven curative modality for hematologic malignancies. The success of unrelated-donor transplantation has been achieved through a better understanding of the immunobiology of the HLA system and through more precise and comprehensive matching of donors and recipients. The extensive polymorphism of HLA genes confers important biological implications affecting engraftment, graft-versus-host disease and overall survival. Although more-complete HLA identity of the donor and recipient is associated with optimal transplant outcome, new information suggests that not every HLA disparity is functionally relevant. Future advances in unrelated-donor transplantation must include the identification of tolerable HLA mismatches, so that more patients may benefit from this therapeutic modality. Furthermore, the role of cytokine-gene polymorphisms and minor histocompatibility genes in transplant outcome requires investigation. Delineation of the function of these markers as transplantation determinants may provide alternative means for optimizing the results of hematopoietic cell transplantation.

On the leachability and sources of some elements in sediments from the Bothnian Sea and the Gotland Deep (the Baltic Sea).

The partitioning of some elements in sediments of two Baltic Sea basins (the Bothnian Sea and the Gotland Deep) was established by leaching experiments. Both partial (dithionite-citrate-bicarbonate) and "total" (HCl-KClO3) leach were used. Trace metals of both natural and anthropogenic sources (Co, Cu, Ni, Pb, Zn) were evaluated for affinity to terrestrial material and to authigenic mineral elements (Fe, S). The results indicate that the easily leachable "mobile" fraction is not necessarily a good indicator for pollution. Longer stagnation periods result in intensive pyrite formation in the Gotland Deep which mask pollution trends. The permanent oxic conditions in the Bothnian Sea may affect a dissolution capability for selected elements.

The biological significance of HLA-DP gene variation in haematopoietic cell transplantation.

Although it has been over 25 years since HLA-DP was mapped to the major histocompatibility complex (MHC), its biological functions remain ill-defined. We sought to test the hypothesis that HLA-DP functions in a manner similar to that of other class II genes by measuring the risk of clinically severe grades III-IV acute graft-vs.-host disease (GVHD) associated with recipient HLA-DP disparity after haematopoietic cell transplantation. HLA-DPB1 exon 2 was sequenced in 205 patients who underwent transplantation from HLA-A, -B, -C, -DRB1 and -DQB1 allele-matched unrelated donors. HLA-DPB1 mismatched recipients experienced a significantly increased risk of acute GVHD compared with HLA-DP-identical transplants. Patients who were mismatched for a single HLA-DPB1 allele had an odds ratio (OR) of 1.0 (0.5, 2.2; P = 0.99) and patients who were mismatched for two alleles had an OR of 2.2 (1.0, 4.9; P = 0.06) for developing acute GVHD. Compared with matched and single-allele mismatched transplants, patients who were mismatched for two DPB1 alleles had an OR of 2.2 (1.2, 4.1; P = 0.01). HLA-DP plays an important role in the alloimmune response. A threshold effect of multiple HLA-DP disparities is evident in determining the risk of acute GVHD after haematopoietic cell transplantation from unrelated donors.

Hypoxia induces hexokinase II gene expression in human lung cell line A549.

During adaptation to hypoxic and hyperoxic conditions, the genes involved in glucose metabolism are upregulated. To probe involvement of the transcription factor hypoxia-induced factor-1 (HIF-1) in hexokinase (HK) II expression in human pulmonary cells, A549 cells and small-airway epithelial cells (SAECs) were exposed to stimuli such as hypoxia, deferoxamine (DFO), and metal ions. The largest increase in HK-II (20-fold for mRNA and 2.5-fold for enzymatic activity) was observed in A549 cells when exposed to DFO. All stimuli selectively increased the 5.5-kb rather than 4-kb transcript in A549 cells. Cycloheximide and actinomycin D inhibited these responses. In addition, cells were transfected with luciferase reporter constructs driven by the full-length HK-II 5'-regulatory region (4.0 kb) or various deletions of that region. A549 cells transfected with the 4.0-kb construct and exposed to hypoxia or DFO increased their luciferase activity 7- and 10-fold, respectively, indicating that HK-II induction is, at least in part, due to increased gene transcription. Sixty percent of the inducible activity of the 4.0-kb construct was shown to reside within the proximal 0.5 kb. Additionally, cotransfection with a stable HIF-1 mutant and the 4.0-kb promoter construct resulted in increased luciferase activity under normoxic conditions. These results strongly suggest that HK-II is selectively regulated in pulmonary cells by a HIF-1-dependent mechanism.

Mouse hexokinase II gene: structure, cDNA, promoter analysis, and expression pattern.

In mammalian tissues, the phosphorylation of intracellular glucose to glucose-6-phosphate (Glu-6-P) is facilitated by four distinct hexokinase (HK) isoenzymes, designated as HKI-IV. Because of the role of HKII as a leading glycolytic enzyme in insulin-sensitive tissues such as skeletal muscle, heart, and adipose tissue, defects in HKII function could contribute to the development of insulin resistance and perhaps Type 2 diabetes. As a first step towards elucidation of the physiological role of HKII in insulin resistance and type 2 diabetes using mouse knock-out models, we determined the genomic structure, sequence of the cDNA and of 4.8 kb of the 5' regulatory region, and tissue-specific expression of the mouse HKII gene. The gene comprises 18 exons that span approximately 50 kb of DNA. Nucleotide sequence of the proximal promoter revealed a number of conserved putative transcription factor binding motifs. We also found numerous repeat elements throughout the mouse HKII gene. The mouse HKII cDNA is approximately 5.5 kb in length and contains an open reading frome of 2751 bp encoding a protein of 917 amino acids. The mouse HKII gene is predominantly expressed in skeletal muscle, heart, and adipose tissue. The transcription initiation and polyadenylation sites for the mouse HKII mRNA were similar to those of the rat and human genes.

Functional consequences of naturally occurring variants of human hexokinase II.

Hexokinase II (HKII) catalyses a key step in glucose metabolism and can be regarded as a candidate gene for insulin resistance and type 2 (non-insulin-dependent) diabetes mellitus. We observed previously four amino acid substitutions among Finnish type 2 diabetic patients: Gln142His, Ala314Val; 0.9%, Arg353Cys; 2.7% and Arg775Gln; 2.7%. The Arg775Gln mutation was also observed in normal control subjects (2.1%) and the Gln142His substitution was found in both Type II diabetic and normal subjects with similar frequencies (approximately 20%). Since Gln at position 142, Ala at 314 and Arg at 775 are present in human and rat hexokinases and could be important for structure and function of the enzyme, we generated all four substitutions by site-directed mutagenesis and expressed them in E.coli. None of these substitutions had any effect on HKII catalytic activity, K(m) or Vmax for glucose values in vitro. Thus unless these substitutions have an impact on enzyme activity or regulation in vivo, it is unlikely that these substitutions contribute to the aetiology of Type II diabetes.

The human hexokinase II gene promoter: functional characterization and detection of variants among patients with NIDDM.

Hexokinase II (HKII) plays an important role in facilitating glucose uptake by skeletal muscle, heart, and adipose tissue in response to insulin. We have cloned and sequenced the proximal promoter region of the human HKII gene, determined the transcription start sites and screened the 2.0 kb of the proximal 5' flanking region for variants in non-insulin-dependent diabetic patients and control subjects. We found three variants in this region, one in the 5' untranslated region (G-->C at +217) and two in the promoter region (T-->G at -1043 and G-->A at -1159). The allele frequencies of these variants did not differ between the diabetic and control subjects and these variants are not associated with insulin resistance. Various segments of the human HKII promoter were tested for driving expression of the luciferase reporter gene. The proximal 500 bp and 400 bp of the promoter were sufficient to drive maximal activity in adipocyte (3T3F442A) and myocyte (C2C12F3) cell lines, respectively. This region of the promoter is GC-rich and contains eight consensus binding sites for the transcription factor Sp-1, five for AP-2, two putative response elements for each of insulin and cyclic AMP. The proximal 175 bases of the promoter retained only 7-15% of maximal activity. Sequence elements located between positions -304 and -215 accounted for approximately 80% of the basal HKII promoter. In addition, the region between -215 and -184 contains a negative regulatory element for expression in 3T3F442A but not in C2C12F3 cells.

Molecular patterns and sequence polymorphisms in the red and green visual pigment genes of Japanese men.

The red-green pigment gene arrays of 203 (101 from a previous study and 102 from this study) randomly selected men of Japanese ancestry from the Seattle area were screened for the abnormal molecular patterns (deletions and red/green or green/red hybrid genes) that are usually associated with defective color vision. Such molecular patterns were found in approximately 5% of these individuals, which is equivalent to the frequency of phenotypic color vision defects in Japanese males in Japan. Thus, the majority of hybrid genes carried by Japanese males appear to be associated with defective color vision. In contrast, the frequency of hybrid genes among Caucasians and African-Americans is approximately two and five times the frequency of color vision defects in these two ethnic groups, respectively. The coding sequences of 50 males of Japanese ancestry were determined. All the polymorphisms in the red and green pigment genes that were detected in the Japanese sample had been observed in Caucasians and African-Americans. The same polymorphisms of the red pigment gene were present in the green pigment gene, suggesting that gene conversion contributes to sequence homogenization between these pigment genes. As is the case for Caucasians, exon 3 of the red and green pigment genes was observed to be a hot spot for recombination and gene conversion. Fewer polymorphic sites (4 vs 11) and haplotypes (5 vs 14) of the red pigment gene were observed in Japanese than in Caucasians. The Japanese population was more uniform with respect to the red pigment gene, with 70% of individuals having the same haplotype, as compared with the 43% for the Caucasian population. This difference was largely due to the lower degree of polymorphism at position 180 of the red pigment gene in Japanese (84% Ser and 16% Ala vs 62% Ser and 38% Ala.) The number of polymorphic sites and haplotypes in the green pigment gene was similar in the two populations. Nevertheless, the Japanese population was more uniform with 65% having the same haplotype. The difference in the frequency of alleles at position 283 accounted for this difference in haplotype distribution.