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This chapter provides the nomenclature for factors of the HLA system, 2002. A number of previously described class I and II gene fragments within the HLA region are named in this system. Official designations are given to these gene fragments. The names LMP2 and LMP7 used previously for the two proteasome genes in the HLA class II region have been renamed by the Human Genome Nomenclature committee (HGNC) as PSMB9 and PSMB8, respectively. This system introduces the additional digit for synonymous variation and all allele names that are currently five digits or above are renamed accordingly. The use of an optional “N’” or “L” suffix to an allele name to indicate either “Null” or “Low” expression was introduced in previous Nomenclature Reports. Three new suffixes are introduced in this system. An “S” to denote an allele specifying a protein that is expressed as a soluble “Secreted” molecule but is not present on the cell surface; a “C” to indicate an allele product that is present in the “Cytoplasm” but not at the cell surface; an “A” to indicate “Aberrant” expression where there is some doubt as to whether a protein is expressed or not. There is evidence of differential splicing of HLA-G that leads to the production of both membrane-bound and soluble forms of the same allele. The IMGT/HLA Sequence Database contains sequences for all HLA alleles officially recognized by the WHO Nomenclature Committee for Factors of the HLA System and, provides users with online tools and facilities for their retrieval and analysis.

Recently a number of new genes have been identified within the HLA region including some whose functions are related to HLA class I and I1 genes. The Committee discussed what its strategy should be for the naming of these and further new Julia G. Bodmer, Steven 6. E. Marsh, Ekkehard D. Albert, Walter F. Bodmer, Ronald E. lontrop, Dominique Charron, Bo Dupant, Henry A. Erlich, Renee Fauchet, Bernard Mach, Wolfgang R. Mayr, Peter Parham, Takehlko Sasazuki, Geziena M. Th. Schreuder, Jack 1. Strominger, Arne Svejgaard and Paul la Terasaki

Correspondence to: Dr Steven G. E. Marsh Anthony Nolan Research Institute Royal Free Hospital Pond Street Hampstead London NW3 2QG UK Tel.: þ442072848321 Fax: þ442072848331 e-mail: marsh@ebi.ac.uk Following the decision to hold their next full meeting after the 14th International Histocompatibility Workshop in 2005, the WHO Nomenclature Committee for Factors of the HLA System has decided to publish an interim report listing updated tables of alleles including those assigned since the publication of the last full report in 2002 (1). The alleles named during the period follow the principles established in previous reports (1–17).

Objective Recently, it has been shown that a homozygous 32 base-pair deletion in the gene encoding CKR-5, a major coreceptor for HIV-1, leads to resistance to infection with HIV-1. We have investigated whether HIV-seropositive individuals who were heterozygous for the CKR-5 deletion had a different course of the disease. Design Thirty-five high-risk HIV-seronegative and 99 HIV-seropositive Danish homosexual men followed form 1985 to 1996 and 37 blood donors were analysed for their CKR-5 genotype by polymerase chain reaction. Results Two (6%) of the 35 HIV-seropositive subjects at high-risk of infection were homozygous and seven (20%) were heterozygous for the CKR-5 deletion. This was not significantly different from the distribution in normal donors. Twenty-two (22%) of the 99 HIV-seropositive subjects were heterozygous and none was homozygous. Two subgroups of patients who had an opposite course of the HIV disease were identified. Of nine long-term non-progressors, six (66%) were heterozygous for the deletion. This frequency is significantly higher than in nine rapid progressors of whom non was heterozygous. The frequency of heterozygotes in long-term non-progressors was also significantly higher than in the cohort as a whole. A Kaplan-Meier plot of the HIV-seropositive subjects, of whom 57 developed AIDS, showed a significantly better prognosis within the first 7 years of follow-up for those who were heterozygous for the deletion. Heterozygous individuals also had a significantly slower decrease in CD4 T-cell count per year. Conclusion Individuals who are heterozygous for the 32-base-pair deletion in the CKR-5 gene have a slower decrease in their CD4 T-cell count and a longer AIDS-free survival than individuals with the wild-type gene for up to 11 years of follow-up.

The multiple sclerosis (MS)-associated HLA major histocompatibility complex (MHC) class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show that together the DRB1 and DRB5 loci may influence susceptibility to MS. We demonstrate that a T cell receptor (TCR) from an MS patient recognized both a DRB1*1501-restricted myelin basic protein (MBP) and DRB5*0101-restricted Epstein-Barr virus (EBV) peptide. Crystal structure determination of the DRB5*0101-EBV peptide complex revealed a marked degree of structural equivalence to the DRB1*1501-MBP peptide complex at the surface presented for TCR recognition. This provides structural evidence for molecular mimicry involving HLA molecules. The structural details suggest an explanation for the preponderance of MHC class II associations in HLA-associated diseases.

Objective: Neutralizing antibodies (NAbs) occur in a proportion of multiple sclerosis (MS) patients treated with interferon (IFN)-β. NAbs impair the effect of treatment. The biological effect of IFN-β can be measured as the induction of the myxovirus resistance protein A (MxA) molecule. However, other markers could be more sensitive for evaluating the response to IFN-β. We used DNA array analysis to identify genes that are strongly induced in blood cells by IFN-β, and measured their expression in MS patients with different NAb levels. Methods: Gene expression was studied on DNA arrays in untreated patients, in NAb negative patients, and in MS patients with varying NAb levels 9–12 h and 36–48 h after IFN-β administration. The expression of selected genes was measured by real-time PCR. NAb levels were assessed by a cytopathic effect assay. Results: Several hundred genes were induced 9–12 h after an injection of IFN-β. The molecules CXCL10, CCL2 and IFI27 were among the most strongly induced. Gene induction was generally much less pronounced after 36–48 h, but IFI27 remained strongly induced. The strong induction of these molecules and MxA was confirmed by real-time PCR. Induction of MxA, CCL2, CXCL10 and IFI27 was reduced in patients with low NAb levels and lost in patients with intermediate/high NAb levels. Conclusion: We identify IFI27, CCL2 and CXCL10 as sensitive biomarkers for the response to IFN-β. The expression of these markers adequately reflects bioactivity of IFN-s as documented by the decreased induction in low NAb-positive patients and the lost induction in patients with moderate/high NAb levels.

A non-selected group of 50 patients with recurrent herpetic keratitis, subclassified into groups of stromal or epithelial forms was typed for HLA-ABC antigens. In none of the groups was a statistically significant association to a certain HLA antigen found. When these data are combined with other reviewed reports on HLA types in recurrent herpetic keratitis, a statistically significant association between HLA-B5 and the whole group of recurrent herpetic keratitis, as well as between the subgroup of recurrent stromal keratitis, could be demonstrated. Recurrent epithelial keratitis showed an insignificant association to this antigen.

A case of iso-immune neonatal thrombocytopenic purpura caused by a maternal, complement fixing, platelet iso-antibody, anti-PlGrLyB1, is reported. The problems concerning the diagnosis of this disease are discussed. Although the PlGrLyB1-antigen is known to be present on both platelets and leukocytes, and though accordingly the mother's blood contained strong leukocyte agglutinins no leukocytopenia was observed in the patient. The reasons for this observation are discussed. No platelet antibody could be demonstrated in the patient's blood, but the maternal antibody belonged to the γG globulins and was thus placenta-permeable. Evidence is presented of a gene dosage effect of the PlGrLyB1-gene among family members.

Thirty-six unrelated Danish patients with pauciarticular Juvenile Chronic Arthritis (PJCA) and 120 controls were typed for HLA-DPw1-w6 and the local specificity CDPHEI with bulk-expanded Primed Lymphocyte Typing (PLT) cells. The frequency of HLA-DPw2 was 52.8% in PJCA patients and 16.7% in controls (relative risk, RR = 4.5; P less than 0.001). The antigens HLA-Dw5 and/or Dw8 were present in 50% of the patients and in 21.3% of the controls (RR = 4.2; p less than 10(-3)). DPw2 was not associated (in linkage disequilibrium) with Dw5/w8 in patients or in controls, and the DP and D associations with PJCA were independent of each other. However, the combined presence of DPw2 and Dw5 and/or Dw8 gave a significantly higher risk of PJCA than each antigen alone indicating interaction of DP and DR gene products. PJCA is the first disease definitely found to be associated with a DP antigen.

HLA typing of an HLA-B/GLO recombinant family using bulk-expanded DP(SB) alloreactive T-cells (GNN1 to 6) as well as conventional HLA-ABC, D, DR. and DQ typing showed that a paternal recombination had taken place between HLA-DR and DQ on the one hand and HLA-DP(SB) on the other. The recombinant child was DPw4, 6-heterozygous and differed in terms of class II determinants only for DPw6 from two otherwise HLA identical siblings, who were probably DPw4/4-homozygous. These siblings did not stimulate the recombinant in MLC whereas they responded to his cells indicating that DPw6 can stimulate in primary MLC. Moreover, it was possible to generate and bulk-expand DPw6-reactive lymphocytes (PLs) between MT and MA as responders and BN as stimulator. The correlation coefficient (r) between the reaction of these PLs and the GNN6 (anti-DPw6) reagents was 1.0 when tested against a panel of 71 individuals. This is the first report demonstrating that the DPw6 gene, like the DPw1-w5 genes, is located between DR/DQ and GLO. The frequency of the DPw6 antigen in 41 unrelated, randomly selected Danes was 7%. HLA-DP typing of the BN family revealed that the DPw6 positive but not DPw6 negative family members gave intermediate responses with one (GNN2B) but not with another (GNN2A) anti-DPw2-reagent. Studies of 63 healthy individuals (unrelated to the BN family) revealed seven similar discrepancies between GNN2A and GNN2B. Strikingly, all four DPw6 positive stimulators gave rise to such discrepancies indicating that the GNN2B reagent is cross-reactive and recognize a common part of the DPw2 and DPw6 molecules. No cases of GNN2A+/GNN2B- stimulators were found.

Investigations of HLA and blood-groups were carried out in 68 patients with infectious mononucleosis comprising all known cases diagnosed within one year in a restricted geographical area of Denmark. The HLA distribution of these patients did not differ significantly from that of controls. Combining the results of the present investigation with two previous studies did not show any significantly different distribution from that of combined control groups. The ABO and Rhesus typing was in accordance with that found in a major Danish control group. However, available studies do not exclude the possibility that HLA-D/DR or still unknown HLA factors may be involved in the susceptibility to mononucleosis.

Lymphocytes from highly selected donors were primed for 10 days and subsequently bulk-expanded in IL 2 (TCGF) containing cultures. Two well-discriminatory PLT (CDP = Copenhagen DP) reagents against each of the DPw1-w6 specificities and one against each of the two “new” specificities, CDP4s and CDPHEI, were selected for further studies. Three combinations made in two recombinant families and four of ten HLA-A, B, and DR compatible combinations discriminated well in contrast to seven of 46 DR compatible, but HLA-A or B incompatible combinations. All reagents gave highly reproducible results, and high correlations (r-values between 0.73–1.00) for DP assignments were obtained with CDP and GNN reagents. No triplets were found for the DPw1 - w6 and CDP HEI specificities. The “new” specificity CDP HEI defined in an HLA-DR/GLO recombinant family gave a coefficient of correlation with GNN 8 of 0.91. Another “new” specificity, CDP4s constitutes a subgroup (“split”) of DPw4. The gene frequencies of DPw1 - w6 estimated in 102 unrelated randomly selected Danes agreed with those reported for other Caucasoid populations. The gene frequencies CDP HEI and CDP4s were 0.03 and 0.08, respectively. The associations between DR3-DPw1, DR2-DPw4, and DRw6-DPw2 were confirmed. It is concluded that DP-typing with bulk-expanded reagents is a reliable and so far the only technique which can reveal the polymorphism of the DP gene products.

A new homozygous typing cell (HTC), SK is described. Mixed leukocyte culture (MLC) showed that SK is homozygous for HLA-D"H" (DB6) defined by the HTC, Herluf, and that the HLA-D typing results obtained by Herluf and SK are highly significantly correlated (Kendall's R = 0.46, p = 2.5 X 10(-5)). Both Herluf and SK are also homozygous for a new class II determinant, DN-1, defined by the monoclonal anti-B-lymphocyte antibody, 9w925, developed by Aizawa. The corresponding DN-1 antigen was present in 2.2% of 136 random, unrelated Danes and in all of six unrelated HLA-D"H" positive but in none of 20 HLA-D"H" negative individuals. Thus, there is an absolute and highly significant (p = 4 X 10(-6)) association between the cellularly defined HLA-D"H" determinant and the serologically defined DN-1 antigen, which strongly suggests that HLA-D"H" can now be detected serologically. DN-1 may be identical to DRw12 which is, however, poorly defined. The HTC-donor SK was immunized by pregnancy, and her serum contains anti-HLA-B7, which can easily be absorbed, and anti-B-lymphocyte antibodies which reacted with cells from 87.7% of 536 unrelated Danes. The reaction pattern of this serum is negatively associated with DR3, w6, and w8. This serum may define a new broad, cross-reacting antigen belonging to the same group as DRw52 and DRw53 or DQw1-w3, but it is clearly different from each of these antigens.

Thirty-three Danish women selected from a prospective study of postpartum thyroiditis were HLA-DR typed. All women had positive titers of antimicrosomal antibodies, and 20 women developed thyroid dysfunction after delivery. DR5 and the phenotype 4.5 were significantly increased in the whole group (p less than 0.001) and strongly associated to hypothyroidism (p less than 0.01), whereas DR3 was insignificantly increased in thyrotoxic women. It is concluded that postpartum hypothyroidism is an autoimmune disorder and may be an early manifestation of Hashimoto's thyroiditis.

A study of four families with a cross-over within the HL—A region and in which the crossover parent is heterozygous at PGM3 indicates with a probability of about 95% that the chromosomal orientation of the three linked loci is LA · FOUR · PGM3.

An MLC test system is described utilizing 105 responding and 105 stimulating lymphocytes in a total of 1 ml of culture medium. Some of the variables of the test have been evaluated and the reproducibility of the test within a given day and between days investigated according to a Workshop protocol. This system was first applied to three groups: HL-A identical siblings, one haplotype different family members and two haplotype different individuals. Within each group, the log converted stimulation ratios are normally distributed, allowing the use of simple statistical analysis in repeated testings. In addition, a group of weakly positive MLC reactions can be detected. This group includes the LD identical but SD different sibling combinations. Within the same group we find some unrelated HL-A sero-identical combinations and the so-called LD typing response, used to identify LD determinants by means of LD and SD homozygous stimulating test cells. It is concluded that this MLC test system allows identification of identical combinations, weakly positive combinations and one and two haplotype responses even within unrelated combinations. This test system may have advantages over the microtiter test system because the large culture volume gives a low concentration of blastogenic factors in typing responses.

We have expressed the human MHC class II molecule, HLA-DRA1*0101/-DRB1*0401 (DRB1 *0401), in Drosophila melanogaster Schneider 2 cells under control of the Drosophila metallothionein promoter. Upon induction with CuSO4, flow cytometry revealed expression of DRBl *0401 on the surface of the Drosophila cells at high levels. The membrane-bound class II molecules could present peptides specifically to DRB1 *0401-restricted T cells. Drosophila-expressed DRBl *0401 molecules revealed a decreased N-linked glycosylation as compared to DRB1 *0401 molecules purified from a human B-cell line. The purified DRB1*0401 molecules from Drosophila cells were dissociated into subunits in SDS-PAGE but could be stabilized with a peptide known to bind DRBl*0401 with a high affinity, indicating that the recombin-ant class II molecules from Drosophila cells are either empty or occupied by low affinity endogenous peptides. This assumption was further substantiated by the observation that the class II molecules from Drosophila cells had a much higher peptide-binding capacity than DRBl*0401 molecules derived from the human B-cell line. Otherwise, the two species of DRB1 *0401 had similar peptide-binding specificities and affinities. The purified recombinant DRB1*0401 molecules also showed biological activity because immobilized complexes of DRB1*0401 and synthetic peptides specifically stimulated DRB1*0401-restricted T cells.

A T-cell receptor's (TCR) recognition of a human leukocyte antigen (HLA)-peptide complex (pHLA) is normally described as being restricted by the HLA molecule and specific for the peptide. This is, however, not always true. Several TCRs have been described, which cross-react with other peptides bound to the restricting HLA molecule. This phenomenon has been considered a variant of molecular mimicry and is suggested to be one of the mechanisms behind autoimmunity. The positive selection of T cells in the thymus imposes low-affinity recognition of the TCRs toward self-pHLA, which increases the probability of the TCR to be promiscuous by nature, and further implies that the T-cell repertoire contains TCRs prone to be autoreactive and thus able to induce autoimmunity. We present an autoimmune TCR showing extreme cross-reactivity to several pHLA comprising both own HLA class II restriction element and allogeneic HLA class II restriction elements in complex with both self-derived and microbially derived peptides. The existence of such a significant cross-reactivity in the context of distinct HLA-DR molecules might be more common among autoimmune TCRs than previously anticipated and potentially reveals a new way of designing altered peptide ligands for therapeutic use.

C1q deficiency is a rare condition associated with a systemic lupus erythematosus (SLE)-like syndrome and recurrent infections. Here we present the molecular basis behind C1q deficiency in three sisters of Inuit origin. Initial examination for complement deficiency showed no function of the classical complement activation pathway in the patients; the lectin and alternative pathways were intact. No C1q or tow molecular weight Clq was detected in sera and no anti-C1q autoantibodies were found. Sequencing of the C1q genes revealed a novel missense mutation (Gly-Arg) in codon 217 of the B chain. All sisters were homozygous for the mutation: both parents were heterozygous. None of 100 healthy controls carried the mutation. Our findings define a third class of molecular mechanisms behind C1q deficiency, where missense mutations cause a lack of detectable C1q-antigen in serum. (c) 2007 Elsevier Inc. ALL rights reserved.

In this study, the results from three Nordic linkage disequilibrium screens in multiple sclerosis (MS) were investigated, in a new sample set of 314 Nordic MS trios from Denmark, Norway, Sweden and Iceland. Among 30 non-HLA and two HLA microsatellite markers individually genotyped, eight markers displayed distorted transmission with uncorrected P-value

Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients. The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant T cells enhance proliferation of the malignant cells in an SE- and MHC class II–dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4+ T-cell lines also enhance proliferation of the malignant cells. The growth-promoting effect depends on direct cell-cell contact and soluble factors such as interleukin-2. In conclusion, we demonstrate that SE triggers a bidirectional cross talk between nonmalignant T cells and malignant CTCL cells that promotes growth of the malignant cells. This represents a novel mechanism by which infections with SE-producing bacteria may contribute to pathogenesis of CTCL.

Cytokines are thought to play an important role in the pathophysiology of graft-versus-host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNCs) from 21 patients with hematologic malignancies and their HLA-identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNCs were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNCs of recipient, donor, or HLA-mismatched third-party origin. The gene expression of interleukin (IL)–2, IL-4, IL-10, IL-18, tumor necrosis factor α, and transforming growth factor β in each culture was then measured by real-time quantitative reverse transcriptase-polymerase chain reaction. The composition of the responder MNCs differed between patients and donors and changed after HCT, with a possible influence on the results. Early after transplantation (day +14), the IL-10 messenger RNA (mRNA) level in response to recipient or donor antigens was higher in patients who did not develop clinically significant acute GVHD when compared with the level in patients who subsequently developed acute GVHD grades II to IV (P = .005 and P = .004, respectively). The IL-10 mRNA level on day +14 was highly correlated with the pretransplantation mRNA level of the recipient MNCs but not with the level of the donor MNCs; this suggests that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNCs obtained before transplantation from recipients whose disease progressed or relapsed after the transplantation when compared with the level in patients whose disease did not progress or relapse (P = .03). In conclusion, a high IL-10 gene expression in the recipient MNCs may be related to a reduced incidence of acute GVHD grades II to IV and a reduced graft-versus-tumor effect after HCT with nonmyeloablative conditioning.

Chimerism analysis is an essential tool in the follow-up of patients after allogeneic stem cell transplantation. High-resolution methods for chimerism analysis based on real-time quantitative polymerase chain reaction (RQ-PCR) with a detection limit of 0.1% marker-specific cells are especially valuable in the detection of patient-derived subpopulations for the monitoring of minimal residual disease. Using artificial chimeric mixtures of genotypically different cells, we optimized and evaluated the intrasample variation, accuracy, and detection limit of chimerism analysis based on RQ-PCR of short insertion and deletion polymorphisms. Furthermore, automated setup by robot was evaluated. The results were accurate, with acceptable intrasample variation at and above 0.1% marker-specific cells. The sensitivity was mainly limited by background values. Chimerism results based on RQ-PCR were similar to results based on PCR of short tandem repeats when samples from recipients of transplants with nonmyeloablative conditioning were analyzed. Furthermore, automated setup was feasible in a time-, labor-, and reagent-conserving manner.

Reduced levels of somatic hypermutation (SHM) have recently been described in IgG-switched immunoglobulin genes in a minority of patients with common variable immunodeficiency (CVID), demonstrating a disruption of the normal linkage between isotype switch and SHM. To see if, irrespective of isotype, there is a tendency to use unmutated immunoglobulin genes in CVID, we studied SHM in κ light-chain transcripts using a VκA27-specific restriction enzyme-based hot-spot mutation assay (IgκREHMA). Hot-spot mutations were found in 48% (median; reference interval, 28%-62%) of transcripts from 53 healthy controls. Values were significantly lower in 31 patients (median, 7.5%; range, 0%-73%; P < .0000001) of whom 24 (77%) had levels below the reference interval. Low levels of SHM correlated with increased frequency of severe respiratory tract infection (SRTI; P < .005), but not with diarrhea (P = .8). Mannose-binding lectin (MBL) deficiency also correlated with SRTI score (P = .009). However, the correlation of SHM and SRTI was also seen when only patients with normal MBL genotypes were analyzed (n = 18, P = .006). A slight decline of mutated fractions over years was noted (P = .01). This suggests that most patients with CVID fail to recruit affinity-maturated B cells, adding a qualitative deficiency to the quantitative deficiency characterizing these patients.

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)-identical sibling donors after non-myeloablative conditioning with fludarabine and total body irradiation. Twenty-seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft-versus-host disease (GVHD) grades II-IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4-151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1-year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP (P = 0.008). With a median follow-up of 602 d, the 2-year estimates for overall survival, progression-free survival, non-relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long-term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year.

The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role.

Investigation of coaffected sib pairs is one method to determine the genetic influence on the clinical presentation of many complex diseases, such as multiple sclerosis (MS). Investigation of the clinical concordance in coaffected sib pairs may be a prerequisite to identify genes that modify the clinical outcome. The aim of this study was to investigate a possible genetic influence on selected demographic and clinical variables among familial Scandinavian MS cases.We identified 136 Caucasian Scandinavian families with MS coaffected sib pairs from Denmark, Norway and Sweden. Cohen's kappa coefficient and the intraclass correlation coefficient were used to assess concordances in sib pairs. Furthermore, clinical features and HLA-DR2 carrier status were compared among the probands of sib pairs.We found significant concordance of the disease course (kappa = 0.28, P0.001) and adjusted age of onset (r = 0.23, P = 0.028). Among probands of sib pairs, HLA-DR2 carrier patients had a younger age of onset (P = 0.024).Analyses of Scandinavian coaffected sib pairs suggest that disease course and age of onset are partly under genetic control. Furthermore, HLA-DR2 in probands of sib pairs suggests importance for age of onset.

We report the first two genome-wide screens for linkage disequilibrium between putative multiple sclerosis (MS) susceptibility genes and genetic markers performed in the genetically homogenous Scandinavian population, using 6000 microsatellite markers and DNA pools of approximately 200 MS cases and 200 controls in each screen. Usable data were achieved from the same 3331 markers in both screens. Nine markers from eight genomic regions (1p33, 3q13, 6p21, 6q14, 7p22, 9p21, 9q21 and Xq22) were identified as potentially associated with MS in both screens.

Summary Several studies indicate genetic involvement of Th2 cytokines in allergic diseases. Interleukin (IL)-13 has been mapped to the cytokine cluster on chromosome 5q31–33, which has been associated with atopic conditions. Recently, an association was reported between the T allele in a promoter polymorphism in the IL-13 gene (C to T exchange) at position −1055 and allergic asthma in a population study in the Netherlands. This observation was apparently confirmed in a case–control study using probands and spouses from a Dutch asthma family study, but the polymorphism in that study was reported to occur at position −1111. In the present study, we established that this polymorphism is located at position −1024 relative to the ATG translation initiation codon, and investigated whether it confers a genetic predisposition to atopic conditions and the Th1 condition multiple sclerosis (MS) in Caucasian subjects. We confirmed the association between the IL-13 −1024TT genoype and inhalation allergy (P = 2.4E-02). By combining the data from the three studies, we demonstrated a strong association (P = 1.09E-05) between the IL-13 −1024 marker and inhalation allergy. Furthermore, we showed for the first time that this association also exists in atopic dermatitis (P = 2.0E-02). No association with MS was found.

Via cytoplasmic signal transduction pathways, cytokines induce a variety of biological responses and modulate the outcome of inflammatory diseases and malignancies. Crohn's disease is a chronic inflammatory bowel disease of unknown etiology. Perturbation of the intestinal cytokine homeostasis is believed to play a pivotal role, but the pathogenesis of Crohn's disease is not fully understood. Here, we study intestinal T cells from Crohn's disease and healthy volunteers. We show that STAT3 and STAT4 are constitutively activated in Crohn's patients but not in healthy volunteers. The activation is specific, because other STAT proteins are not constitutively activated. Furthermore, the STAT3 regulated protein, SOCS3, is also constitutively expressed in Crohn's patients but not in healthy volunteers. Taken together, these data provide evidence of abnormal STAT/SOCS signaling in Crohn's disease. This aberrant activation, so far noted only in malignant cells, establish a new critical approach for better understanding the immunopathogenesis of Crohn's disease.

Udgivelsesdato: 2003-Jan-15 The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)-infected patients with hemophilia. One patient (0.6%) had the CCR5Delta32/CCR5Delta32 genotype (which occurs in approximately 2% of the Scandinavian population) and a rapid disease course. His HIV V3 region contained genotypic features attributable to X4 virus and resembled functionally verified X4 virus and virus from patients treated with a CD4 cell-stimulating drug, tucaresol. Age-related differences in disease progression rate and survival time were seen for CCR5/CCR5 patients. Surprisingly, no protective effect of the CCR5/CCR5Delta32 genotype on disease progression or survival was seen for children but was evident for adults. Age group-related immunologic differences might explain this variation, and transmission route and/or viral phenotype variation within donor virus may be related to the limited protection of the CCR5Delta32/CCR5Delta32 genotype. Sequence comparisons indicate that X4 virus can be selected in vivo due to either absence of CCR5 receptors or relative increase of CXCR4 receptors.

Genetic factors influence susceptibility to multiple sclerosis but the responsible genes remain largely undefined, association with MHC class II alleles being the only established genetic feature of the disease. The Nordic countries have a high prevalence of multiple sclerosis, and to further explore the genetic background of the disease, we have carried out a genome-wide screen for linkage in 136 sibling-pairs with multiple sclerosis from Denmark, Finland, Norway and Sweden by typing 399 microsatellite markers. Seventeen regions where the lod score exceeds the nominal 5% significance threshold (0.7) were identified-1q11-24, 2q24-32, 3p26.3, 3q21.1, 4q12, 6p25.3, 6p21-22, 6q21, 9q34.3, 10p15, 10p12-13, 11p15.5, 12q21.3, 16p13.3, 17q25.3, 22q12-13 and Xp22.3. Although none of these regions reaches the level of genome-wide significance, the number observed exceeds the 10 that would be expected by chance alone. Our results significantly add to the growing body of linkage data relating to multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology. Nitric oxide (NO) is a free radical that participates in a variety of biological processes. It is an important mediator in the immune response. Several studies indicate involvement of NO in the pathogenesis of MS. We studied five markers within the three NO synthase genes with regards to susceptibility and disease course in 156 affected sib-pairs and in 96 “benign” and 96 “severe” definite MS patients and 148 controls. We found no significant association or evidence for linkage in our data sets.

The T cell specific adapter protein (TSAd) encoded by the SH2D2A gene is involved in the control of T cell activation. The gene is located in the 1q21 region, which has been implicated in susceptibility to experimental allergic encephalomyelitis in the mouse. We therefore evaluated whether a polymorphic GA repeat (GA(13)-GA(33)) within the promoter region of the SH2D2A gene shows association to multiple sclerosis (MS). The frequency of the short alleles GA(13-16) was increased among 313 Norwegian MS patients compared to 277 healthy controls (0.332 vs 0.249, OR 1.5, Pc = 0.03). Transmission disequilibrium analysis in 146 Scandinavian families with at least two affected sibs showed increased transmission of GA(16) to MS patients. No linkage or association of MS to four genetic markers flanking the SH2D2A gene was observed. After activation of naive CD4(+) T cells, T cells homozygous for MS associated short alleles displayed lower level of TSAd ex vivo than T cells carrying at least one long allele, which were not associated to MS. Since the SH2D2A protein modulates T cell activation, this may be a mechanism for how short SH2D2A alleles confer susceptibility to develop MS.

The proinflammatory cytokine interferon (IFN)-gamma has been shown to influence the course of multiple sclerosis (MS). The IFN-gamma (IFNG) contains a multiallelic dinucleotide repeat in intron 1. To investigate whether alleles at this locus influence susceptibility to MS, we performed linkage and familial association analyses on 100 sibling pairs from four Nordic countries, and case-control association analysis on 220 intermediately disabled sporadic MS patients and 266 controls. To determine the effect of the polymorphism on disease outcome, we compared genotype frequencies in the most and least disabled octiles of a total cohort of 913 cases. We also measured IFN-gamma mRNA levels in unstimulated peripheral blood mononuclear cells from 46 MS patients and 27 controls grouped according to IFNG intron 1 genotype. Both nonparametric linkage analysis and transmission disequilibrium testing of the 100 sibling pairs produced negative results. Genotype frequencies for intermediate-MS patients did not differ significantly from those for controls; nor did genotype frequencies in the benign-MS octile differ significantly from those in the severe-MS octle. Comparison of IFN-gamma mRNA levels in genotype-conditioned subgroups revealed no significant differences. Thus, alleles at the IFNG intron 1 dinucleotide repeat appear to affect neither MS susceptibility and severity nor IFN-gamma mRNA expression in vivo.

Most prognostic factors in childhood acute lymphoblastic leukemia (ALL) are informative for groups of patients, whereas new approaches are needed to predict the efficacy of chemotherapy for the individual patient. The residual leukemia following 4 weeks of induction therapy with prednisolone, vincristine, doxorubicin and i.t. methotrexate and the in vitro resistance to prednisolone, vincristine, and doxorubicin were measured in 30 boys and 12 girls with B (n = 34) or T lineage (n = 8) ALL. The residual leukemia was quantified after 2 (MRD-D15, n = 29) and 4 weeks (MRD-PI, n = 42) of induction therapy with a precise and reproducible clone-specific PCR technique. The median MRD-D15 and MRD-PI were 0.50% (75% range 0.008-8.1%) and 0.014% (75% range 0.001-2.0%), respectively, and these levels correlated significantly (n = 29, rs = 0.75, P < 0.001). Both the MRD-D15 and the MRD-PI were related to the age of the patient (MRD-D15: rs = 0.48, P = 0.009; MRD-PI: rs = 0.45, P = 0.003). Patients with T lineage ALL had higher MRD-PI than those with B lineage ALL (median MRD-PI: 0.5% vs 0.01%, P = 0.05). The median LC50 (concentration lethal to 50% of cells) for prednisolone was 2.3 μg/ml (75% range 0.05-668). Both MRD-D15 and MRD-PI correlated significantly with the in vitro resistance to prednisolone (MRD-D15: rs = 0.41, P = 0.03; MRD-PI: rs = 0.39, P = 0.01); but not to in vitro vincristine or doxorubicin resistance. The correlations between MRD and in vitro prednisolone resistance were even more pronounced when B cell precursor and T cell leukemia were analyzed separately (B cell precursor ALL: MRD-PI vs prednisolone LC50: n = 33, rs = 0.47, P = 0.006; T cell ALL: MRD-PI vs prednisolone resistance: n = 8, rs = 0.84, P = 0.009). After a median follow-up of 5.0 years (75% range 3.2-6.9) eight patients have relapsed. All of the 21 patients with a MRD-PI ≤0.5% and a prednisolone LC50 ≤10 μg/ml have remained in remission whereas the 7 year event-free survival for the remaining 20 patients was 0.45 ± 0.16 (P = 0.002) Prospective studies in childhood ALL are needed to clarify whether combined monitoring of in vitro drug resistance and residual leukemia early during chemotherapy could offer new ways to classify patients and stratify the intensity of therapy.

To date, four genome screens have been completed in the demyelinating autoimmune disease multiple sclerosis (MS). Although these screens failed to identify any loci with major effects on susceptibility, several novel regions of potential linkage were suggested, including the long arm of chromosome 17. In order to further pursue this promising region we have investigated six highly polymorphic microsatellite markers in 115 Scandinavian families with MS affected sib pairs. Multipoint linkage analysis revealed a peak maximum likelihood score (MLS) of 0.9 in the region of marker D17S787. Stratifying the results on the basis of HLA-DR2 status showed that the linkage was not limited to families segregating for the HLA-DR2 allele as has previously been suggested. In conclusion, our results further support the proposal that a multiple sclerosis susceptibility locus is contained on chromosome 17q.

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon γ (IFN-γ)–inducible protein 10 (γIP-10) and monokine induced by IFN-γ (Mig). We report the novel finding that CXCR3 is also expressed on CD34+ hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34+ progenitors. Freshly isolated CD34+progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase–polymerase chain reaction technique. γIP-10 and Mig induced chemotaxis of GM-CSF–stimulated CD34+ progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block γIP-10–induced and Mig-induced CD34+ progenitor chemotaxis. These chemotactic attracted CD34+ progenitors are colony-forming units—granulocyte-macrophage. γIP-10 and Mig also induced GM-CSF–stimulated CD34+ progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of γIP-10 and Mig but not of chemokine stromal cell–derived factor 1α. γIP-10–induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF–stimulated CD34+progenitors. Moreover, γIP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF–stimulated CD34+progenitors. These results indicate that CXCR3–γIP-10 and CXCR3–Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34+ hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34+ hematopoietic progenitors.

During the development of a sequencing based typing (polymerase chain reaction (PCR)-SBT) protocol for the HLA-A locus, the presence of two bands in addition to the expected full-length product were observed in the template generating PCR in some of the samples investigated. Despite a profound optimisation of the PCR, these new products of lower molecular weight remained present. The new products were not associated with specific alleles. However, the affected samples consisted of peripheral blood lymphocytes isolated from four patients with colorectal cancer, one patient diagnosed with myeloma, and a colon tumor cell line. In order to elucidate the nature of this phenomenon, a number of these products were cloned and sequenced. Sequence analysis revealed that alternative splicing of the HLA-A transcript was responsible for the generation of these smaller products. Thus, a number of clones were generated from transcripts in which a 276 base pair region corresponding exactly to exon 3 had been spliced out. Three additional transcripts lacking exons 2 and 3, exons 3 and 4, and transcripts lacking exons 2-4 respectively, were identified as well. These results are in some respects similar to observations made for HLA-G, one of the nonclassical class I loci, in which a number of alternatively spliced transcripts have been identified. However, no specific functions have been ascribed to these molecules nor have the truncated proteins encoded by these transcripts been identified, thereby questioning the biological significance of these observations. Nevertheless, our findings indicate that alternative splicing of the HLA-A transcript may take place to a small extent in virtually all cells, and it is possible that their generation promote escape from immune surveillance.

In the genetically homogeneous Scandinavian population, we have investigated chromosome 5 and the HLA (human leukocyte antigen) region on chromosome 6p21 by applying linkage and association analyses on 106 white sibling pair families with multiple sclerosis. The importance of genes within the HLA region for the susceptibility of multiple sclerosis has previously been reported. More recently, findings have suggested importance of regions on chromosome 5. Half of chromosome 5 was analyzed by using 14 microsatellite markers and a susceptibility region with a maximum LOD score of 1.1 was identified. Chromosome 6 was analyzed by HLA-DR typing and using the TNF alpha microsatellite marker. A peak maximum LOD score of 2.0 was found at the HLA-DR marker. Association studies were made for all the markers, comparing 106 probands from the sibling pairs with 100 unrelated controls. None of the markers on chromosome 5 showed significant association with multiple sclerosis, whereas strong association between multiple sclerosis and DR2 was found, with an odds ratio of 3.7 (p < 10(-5)). It is surprising that association was not seen for any of the TNF alpha alleles including the 121-bp allele, although this allele was in positive linkage disequilibrium with DR2 in both patients and controls. Our results support the existence of multiple sclerosis susceptibility loci on chromosomes 5p and 6p21.

Mannose-binding lectin (MBL) is a key factor in innate immunity, and lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Accordingly, we investigated whether MBL variant alleles, which are associated with recurrent infections, might be risk factors for CF patients. In 149 CF patients, different MBL genotypes were compared with respect to lung function, microbiology, and survival to end-stage CF (death or lung transplantation). The lung function was significantly reduced in carriers of MBL variant alleles when compared with normal homozygotes. The negative impact of variant alleles on lung function was especially confined to patients with chronic Pseudomonas aeruginosa infection. Burkholderia cepacia infection was significantly more frequent in carriers of variant alleles than in homozygotes. The risk of end-stage CF among carriers of variant alleles increased 3-fold, and the survival time decreased over a 10-year follow-up period. Moreover, by using a modified life table analysis, we estimated that the predicted age of survival was reduced by 8 years in variant allele carriers when compared with normal homozygotes. Presence of MBL variant alleles is therefore associated with poor prognosis and early death in patients with CF. J. Clin. Invest. 104:431-437 (1999).

A combined analysis of data available in the literature has demonstrated that the strongest association in rheumatoid arthritis (RA) is with DR genes rather than DQ or DP genes. Functional and structural data of RA-associated DR molecules suggest that selective binding of peptides is the molecular basis for this association. The establishment of functional transgenic mice expressing RA-associated HLA class II molecules has proven to be useful in the delineation of the role of these molecules in immune responses possibly related to RA and in the development of humanized models for this disease. Such humanized mice develop arthritis upon immunization with type II collagen (CII), which shows similarities with RA. Interestingly, the immunodominant T-cell determinant in CII is derived from positions 261-273, which overlap with a previously identified CII T-cell epitope restricted by the mouse Aq molecule, which is associated with collagen-induced arthritis. Studies in collagen transgenic mice have shown that recognition of this peptide may lead either to T-cell tolerance or to an arthritogenic response. It is therefore proposed that the T-cell recognition of the CII peptide bound by DR molecules is one of the molecular interactions of critical importance in the development of RA and accordingly also an important target for prevention and treatment of this disease.