Association of MICA-TM and MICB C1_2_A microsatellite polymorphisms with tumor progression in patients with colorectal cancer.

PURPOSE: The major histocompatibility complex class I related A (MICA) and MICB molecules are ligands of NKG2D receptors on natural killer cells, gamma/delta T cells, and CD8ass T cells that mediate host antitumor immune response. The role of MICA-TM and MICB C1_2_A alleles in patients with colorectal cancer has not yet been investigated. METHODS: We have analyzed the MICA-TM and MICB C1_2_A polymorphisms in colorectal cancer patients (n = 79) by polymerase chain reaction amplification, subsequent electrophoresis, and sequencing in comparison to a previously analyzed cohort of healthy controls (n = 306). Allele frequencies obtained for MICA-TM and MICB C1_2_A were compared to histopathological data regarding tumor invasion, disease progression, microsatellite instability, and the presence of KRAS mutations (codon 12) and analyzed for possible impact on tumor-related survival (n = 61). RESULTS: Allele frequencies of MICA-TM and MICB C1_2_A polymorphisms were not different in patients with colorectal cancer in comparison to normal controls. In colorectal cancer patients, MICA-TM A4 allele was directly and MICA-TM A5 allele was inversely associated with lymph node involvement and advanced UICC stages. Tumor-related survival in colorectal cancer patients was significantly reduced in the presence of the MICA-TM A4 allele (p = 0.015). In patients with microsatellite stable tumors, survival was reduced in association with the MICA-TM A4 allele (p = 0.006) and MICA-TM A9 allele (p = 0.034), but increased in patients showing the MICA-TM A5 allele (p = 0.042). CONCLUSIONS: Specific MICA-TM alleles seem to influence tumor progression and midterm survival of patients with colorectal cancer, indicating an important role of host innate immune predisposition involving NKG2D mediated antitumor response.

Binding of myelin basic protein peptides to human histocompatibility leukocyte antigen class II molecules and their recognition by T cells from multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease in which myelin proteins have been implicated as autoantigens recognized by pathogenic autoreactive T cells. To study the relationship between human myelin basic protein (hMBP) and HLA alleles associated to MS susceptibility, such as DRB1*1501, the binding of synthetic peptides spanning the entire hMBP sequence to 10 purified HLA-DR molecules was determined. All the hMBP peptides tested showed binding affinity for at least one of the DR molecules analyzed, but three hMBP peptides, included in sequences 13-32, 84-103, and 144-163 were found capable of binding to three or more DR molecules. The hMBP peptide 84-103 was the most degenerate in binding, in that it bound to 9 out of 10 DR molecules tested. Interestingly, it bound with highest affinity to DRB1*1501 molecules. To correlate the binding pattern of hMBP peptides to HLA class II molecules with their recognition by T cells, 61 hMBP-specific T cell lines (TCL) were established from the peripheral blood of 20 MS patients, who were homozygous, heterozygous, or negative for DRB1*1501. Analysis of hMBP epitopes recognized by these TCL and their HLA restriction demonstrated a very good correlation between binding data and T cell proliferation to hMBP peptides. Although virtually all hMBP peptides tested could be recognized by at least one TCL from MS patients, three immunodominant T cell epitopes were apparent among the TCL examined, corresponding exactly to the hMBP peptides capable of binding to several DR molecules. No major difference could be detected in the recognition of immunodominant hMBP peptides by TCL from DRB1*1501 positive or negative MS patients. These results have implications for the role of hMBP as relevant autoantigen, and of DRB1*1501 as susceptibility allele in MS.

Immunoprinting: various genes are associated with increased risk to develop rheumatoid arthritis in different groups of adult patients.

To identify genes that contribute to the manifestation of rheumatoid arthritis we performed association studies via microsatellite analyses of immunorelevant loci (HLA-DRB, 5 T cell receptor loci, TNFa IL1, IL2, IL5R and CD40L). A total of 183 patients and 275 healthy controls were typed in terms of HLA and grouped according to the known predisposing HLA-DRB1 genes (DRB1*04; relative risk approx. 5; DRB1*01, relative risk approx. 2; a third group carried neither allele). Microsatellite polymorphisms characterizing the TCRBV6S3, CD3D, IL1A, IL2, and IL5R genes did not show significant associations with rheumatoid arthritis, whereas TCRBV6S1, TCRBV6S7, TNFa, and CD40L genes may influence relative protection or risk in certain groups of patients. Analysis of a microsatellite marker adjacent to the transcription element alpha (TEA) in the T cell receptor alpha delta complex indicates that in the cohort carrying neither the DRB1*04 nor the DRB1*01 allele the relative risk to acquire rheumatoid arthritis is increased (> 13) or decreased (< 0.07), depending on the inherited microsatellite allele adjacent to the TEA locus. Sequence analysis of the closely linked TEA region from patients and controls revealed a novel dimorphism. Only the newly identified TEA allele leads to binding of a nuclear protein that may be involved in the regulated expression of the TCRDA genes. Subsequent typing of rheumatoid arthritis patients and controls revealed, however, that the association of the microsatellite marker is largely independent of the TEA allele, confirming incomplete linkage in the 2 kb region of the TCRDA locus. These results are discussed in the context of hot spots of recombination in this genomic region and other linked candidate sequences that predispose to develop rheumatoid arthritis.

Predictive value of human leukocyte antigen class II typing for the development of islet autoantibodies and insulin-dependent diabetes postpartum in women with gestational diabetes.

Gestational diabetes mellitus (GDM) is a risk factor for the development of insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus postpartum. To evaluate whether there is any association of human leukocyte antigen (HLA) class II alleles (DR and DQ) with GDM and the postpartum development of IDDM, we analyzed 184 women with GDM from Germany for HLA class II alleles, islet autoantibodies [islet cell autoantibodies (ICA), glutamic acid decarboxylase autoantibodies (GADA), and protein tyrosine phosphatase IA-2 autoantibodies (IA-2A), and the postpartum development of diabetes. No elevation in the frequency of any HLA class II alleles was observed in GDM patients compared to 254 nondiabetic unrelated subjects. DR3 allele frequency was significantly increased in 43 women with islet autoantibodies [corrected P value (Pc) = 0.02], in particular in those with GADA (Pc = 0.002), or in the 24 women who developed IDDM postpartum (Pc = 0.005). In women with GADA, DR4 and DQB1*0302 were significantly elevated (Pc = 0.009). Twenty-five (59.5%) islet antibody-positive women and 17 (74%) women who developed IDDM postpartum had a DR3- or DR4-containing genotype. The cumulative risk to develop IDDM within 2 yr postpartum in GDM women with either DR3 or DR4 was 22% compared to 7% in women without those alleles (P = 0.02) and rose to 50% in the DR3- or DR4-positive women who had required insulin during pregnancy (P = 0.006). Combining the determination of susceptible HLA alleles (DR3, DR4) with islet autoantibody measurement increased the sensitivity of identifying GDM women developing postpartum IDDM to 92%, but did not improve risk assessment above that achieved using GADA measurement alone, which was the strongest predictor of IDDM. These results indicate that women with GDM who have islet autoantibodies at delivery or develop IDDM postpartum have HLA alleles typical of late-onset type 1 diabetes, and that both HLA typing and islet antibodies can predict the development of postpartum IDDM.

Genetic differences between the salt-wasting, simple virilizing, and nonclassical types of congenital adrenal hyperplasia.

Human leukocyte antigen (HLA) alleles and plasma 17-hydroxyprogesterone levels after ACTH stimulation were studied in 134 German families of patients with the salt-wasting (SW), simple virilizing (SV), or nonclassical (NC) late-onset form of congenital adrenal hyperplasia (CAH). Unexpected hormonal evidence for CAH was found in 6 otherwise healthy members of the relatives' group, who, therefore, were considered to be NC cryptic cases. HLA typing revealed a genetic difference between the 2 classical disease forms; SW CAH was strongly associated with Bw47, whereas SV CAH was closely linked to B5(w51). It also confirmed the nearly complete connection of NC CAH with B14. These alleles, especially Bw47 and B14, are mostly components of normally rare haplotypes: A3,Bw47,DR7 and Aw33,B14,DR1, respectively. They do not occur in the families' disease-unaffected haplotypes. Thus, it may be that all or almost all individuals from the general population bearing 1 of them are in fact CAH heterozygotes. Moreover, it seems possible to predict the severity of an infant's disease from his genomic type. The HLA linkage data were consistent with those obtained from ACTH testing, which showed significantly higher 17-hydroxyprogesterone increases in the genetically defined heterozygous relatives of SW patients than in the respective members of SV families. Of the families, 2 were also informative for mapping of the CAH disease gene(s) within the HLA-B to Glo interval.

In vivo and in vitro immune variables in patients with narcolepsy and HLA-DR2 matched controls.

We investigated cytokine levels (interleukin [IL]-1beta, IL-1ra, IL-2, IL-6, tumor necrosis factor [TNF]-alpha, TNF-beta) in plasma and secreted by mitogen-stimulated blood monocytes and lymphocytes; T-cell subsets; and natural killer cell activity in patients with narcolepsy and in human leukocyte antigen (HLA)-DR2 matched controls. The only significant finding was higher IL-6 secretion by monocytes of patients than by those of the HLA-DR2-positive controls. In conclusion, we found no major abnormalities of T-cell function in patients with narcolepsy, but slight alterations of monocyte function deserving further investigation.

Susceptible and protective major histocompatibility complex class II alleles in early-onset pauciarticular juvenile chronic arthritis.

Oligonucleotide typing for alleles of the MHC loci DRB1, DQA1, and DQB1 was performed in 160 patients suffering from EOPA, JCA (or JRA = juvenile rheumatoid arthritis). Allele and haplotype frequencies of the patients were compared with the data of an unrelated healthy control group consisting of 200 individuals. Analysis of frequencies shows that HLA alleles are associated not only with susceptibility to EOPA-JCA but also with protection from the disease. The presence of protection connected with certain HLA alleles was assessed using a calculation which takes into account the condition that if one allele is increased, all other alleles of the same locus must be decreased in compensation. Protection can be assumed only in cases where a given allele has an observed frequency which is significantly beyond the expected compensatory decrease. Thus a hierarchy of associations was observed in EOPA-JCA patients. The alleles of the haplotypes DRB1*11 (12)-DQA1*0501-DQB1*0301 as well as DRB1*08-DQA1*0401-DQB1*0402 were found to be associated with susceptibility to disease, whereas the alleles DRB1*07 and DQA1*0201 converge with significant protection from the disease. Whereas the association with disease susceptibility seems to depend on a sequence motif encoded in certain DQA1 alleles, protection is associated either with alleles of DRB1 or DQA1.

A comprehensive search for segregation distortion in HLA.

Segregation distortion, the non-Mendelian segregation of gametes, has been well documented among diverse groups of organisms. These cases are characterized by extreme segregation ratios found only in males. Previous reports have suggested the existence of segregation distortion operating in the HLA system of humans, a tightly linked complex of genes which regulates the immune system. In mice, some alleles of the T/t complex, which is linked to H-2 (the HLA homologue of mice), cause extreme segregation distortion in wild mice populations. Here we report on the examination of a large body of pedigree data on non-diseased families, scored for the alleles of five HLA region loci. We searched for segregation distortion on the basis of five different models of inheritance: allelic, haplotypic, genotypic, diffuse occurrence in families, and autosomal effects on the sex ratio. There was no clear evidence for segregation distortion. In particular, the possibility of extreme levels of segregation distortion was firmly rejected in the populations examined, thus reducing the likelihood of common distortion-causing HLA associated haplotypes in our species.

A deletion in the second exon of an HLA-DRB1 allele found in a DR2-negative narcolepsy patient.

In this report, we describe a new allele of the HLA-DRB 1 gene carrying a form of mutation that has not been observed before. It appeared in an HLA-DR2-negative narcolepsy patient who, besides HLA-DR4, revealed a serologic HLA-DR blank segregating with HLA-DQ1. Oligotyping showed that the new allele belongs to the HLA-DR8 group. Restriction analysis and DNA sequencing revealed the deletion of 12 bp as well as the substitution of 9 flanking base pairs between codons 36 and 43. The expression of the mutated gene was demonstrated by the presence of its messenger RNA and a few positive reactions with DR8 sera. Without interrupting the reading frame, the mutation leads to a gene product composed of a modified amino acid sequence. We anticipate that the mutation influences the conformation of the molecule with possible consequences concerning immune response.

Identification of two major HLA-B44 subtypes and a novel B44 sequence (B*4404). Oligotyping and solid phase sequencing of polymerase chain reaction products.

PCR-based analyses were performed for the identification of HLA-B44 subgroups. Genomic DNA from six homozygous cell lines and 44 healthy individuals who had serologically tested positive for HLA-B44 was investigated for polymorphism in exons 2 and 3 of the HLA-B44 genes. Two primers were designed for specific amplification of the B*4401 allele in exon 2. None of the tested genomic DNAs, including the cell line "BAU-J" from which the sequence of B*4401 was derived, was amplified successfully using these primers, indicating that the B*4401 sequence may not be correct in position 242-244. For identification of the B*4402 and *4403 subtypes we specifically amplified the B44 gene in exon 3 using two sequence-specific primers. The PCR products, which were obtained from all B44-positive samples (n = 50) and from none of the B44-negative controls (n = 20), were subsequently hybridized with the dig-ddUTP-labeled oligonucleotides. The base substitution at position 146, as described previously for B*4401 and *4402 (C for G), could not be confirmed by oligonucleotide hybridization. In contrast, the oligonucleotide typing for G in position 146 gave positive signals in all B44-positive samples. Except for one, HLA-B44-positive DNAs from LCLs and healthy individuals could be divided into two subgroups according to the polymorphic region in position 195-197. Out of 44 unrelated individuals with B44, 27 (61%) were positive for B*4402 and 16 (36%) were positive for B*4403.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonradioactive HLA class II typing using polymerase chain reaction and digoxigenin-11-2'-3'-dideoxy-uridinetriphosphate-labeled oligonucleotide probes.

We describe a new, simple, rapid, and sensitive nonradioactive technique for the analysis of genetic variations. Genomic DNA was amplified using polymerase chain reaction and amplified DNA was hybridized, with digoxigenin (DIG)-labeled sequence-specific oligonucleotides. High specificity and sensitivity was achieved when labeling the sequence-specific oligonucleotide at the 3' end with only one DIG using digoxigenin-11-2',3'-dideoxy-uridine-5'-triphosphate and DNA deoxynucleotidylexotransferase. The hybridized probes were detected using antidigoxigenin alkaline phosphatase, fab fragments, and X-phosphate/NBT for visualization. This method was applied to the analysis of HLA-DR4-DRB1 alleles in polymerase chain reaction-amplified genomic DNA and resulted in highly specific and sensitive hybridization signals discriminating even in cases of a one-base-pair mismatch. This technique is particularly suited for HLA oligotyping because it allows the use of tetramethylammonium chloride for the simplification of hybridization and washing conditions.

Genetics of insulin dependent diabetes mellitus in Israel: population and family study.

The association between insulin dependent diabetes mellitus (IDDM) and the HLA system was studied in two groups of Jewish patients: 50 Ashkenazim and 42 non-Ashkenazim. The pattern of association of HLA-A and B locus antigens was somewhat different from that observed in European Caucasian patients. HLA-B8 had a higher frequency; B15 and Cw3 were rare in the population studied and were less frequent in IDDM patients than in controls. On the other hand, the frequency of A26, B18, and Bw38 was increased in Ashkenazi patients, but not in non-Ashkenazim, who in turn showed an increase for Bw51. Although the association between IDDM and HLA-A and B locus antigens shows a marked variability in different populations, the association with HLA-DR3 and DR4 is constant feature. There was a typical excess of DR3/DR4 heterozygotes in both patient groups. This heterozygote type carries the highest relative risk, followed by DR4/DR4 homozygotes. These data can well be interpreted by a model of two different HLA-linked susceptibility genes, one associated with DR3 and the other one with DR4, that interact so that different genotypes are associated with different levels of penetrance. This model received further support from studies in 15 multiple case families where there is an excess of affected sib pairs sharing two DR antigens.

Characterization of 4AOHW cell line panel including new data for the 10IHW panel.

There will be a continuing need for well characterized panels of EBV-transformed lymphoblastoid cell lines. Selection of the 4AOH panel was based on prior MHC typing and was intended to ensure representation of ancestral haplotypes from various racial groups. Cells from nonhuman primates, bone marrow donor-recipient pairs, and patients with IDDM were included. Selected cells from the 10IHW were included to enable further characterization. Cells were distributed to participants in the 4AOHW and were typed at multiple loci by a variety of procedures. Non-HLA genes such as TNF were included. Since the cells were distributed "blind" with hidden replicates, it was possible to evaluate the quality of the typing data. An approach to data management is described. The best current estimates of the typing of these cells are presented. The panel will be useful since it provides standards for most alleles at most loci. Since the cells are so well characterized, they represent a useful resource for MHC sequencing and for the evaluation of new typing procedures.

DR2-positive monozygotic twins discordant for narcolepsy.

Narcolepsy runs in families, and recent research has revealed the human leukocyte antigen (HLA) DR2 to be a genetic marker closely associated with the disease. But, as indicated by family studies, other factors contribute to the pathogenesis of narcolepsy. The investigation of monozygotic twins is the most specific research tool for distinguishing between a multigenetic and a multifactorial pathogenetic model. We present clinical and sleep polygraphic data from two pairs of monozygotic twins, and in addition, from some of their first-degree relatives. In both pairs only one twin suffered from the clinical symptoms of narcolepsy/cataplexy. Only in these subjects did night sleep recordings and a multiple sleep latency test reveal both multiple sleep onset rapid-eye-movement periods (SOREMPs) and short mean sleep onset latencies. However, in two of the asymptomatic, HLA DR2+ relatives, short mean sleep onset latencies during the multiple sleep latency test (MSLT) were observed, and one, HLA DR2- relative showed REM sleep two times during the MSLT. Our results strongly favor a multifactorial pathogenetic model for narcolepsy.

Heterogeneity of HLA-D(DR)4 detected by primary and secondary mixed-lymphocyte-culture reactions and by individual HLA-DR4 antisera.

One of our panel families (Sb), in which the paternal haplotypes Dw4, DR4 (a) and Dwblank, DR4 (b) segregate, was tested in primary mixed lymphocyte culture (1.MLC) and in the primed lymphocyte test (PLT). In the 1.MLC, cells which carry the a haplotype strongly stimulate b-haplotype cells, and vice versa. For the PLT, lymphocytes of two family-members were primed against the a haplotype and two against the b haplotype. A strong positive restimulation (RR greater than or equal to 60%) occurred only with cells bearing the original stimulating haplotype. The PLs were tested later against families St and Sm, which possess DR4 haplotypes, and against a panel of 73 unrelated persons. The results show heterogeneity of D(DR)4, suggesting at least three different subgroups: D(DR)4a, present on DR4 cells which strongly restimulate the anti-a PLs; D(DR)4b, on DR4 cells which strongly restimulate the anti-b PLs, and D(DR)4c, on the DR4 cells, which do not restimulate any of the PLs tested here. It seems also possible to differentiate between these subgroups with conventional DR-serology, as the 8W sera 903 and 981 react only with a-haplotype cells of family Sb, and ths 8W sera 872 and 1045 react only with b-haplotype cells.

Different gene defects in the salt-wasting (SW), simple virilizing (SV), and nonclassical (NC) types of congenital adrenal hyperplasia (CAH).

HLA (human leucocyte antigens) alleles and plasma 17-hydroxyprogesterone levels after ACTH stimulation were studied in 134 German families of patients with salt-wasting (SW), simple virilizing (SV), and nonclassical (NC) late-onset forms of CAH. HLA typing revealed a genetic difference between the two classical disease forms. SW-CAH was strongly associated with Bw47 and SV-CAH was closely linked to B5. The nearly complete connection of NC-CAH with B14 was confirmed. Bw47 and B14 were mostly components of the normally rare haplotypes A3, Bw47, DR7 and Aw33, B14, DR1, respectively. They did not occur in the families' disease-unaffected haplotypes. The HLA linkage data were consistent with those obtained from the ACTH stimulation test which showed a higher 17-hydroxyprogesterone increase in the group of genetically defined heterozygous relatives of SW patients than in the groups of heterozygous members of SV and NC families.

Immunogenetic aspects of juvenile chronic arthritis.

The HLA associations of Juvenile Chronic Arthritis are reviewed in the light of the newest results. The most convincing data are available about the early onset pauciarticular Juvenile Chronic Arthritis (EOPA-JCA), where there are highly significant associations with 3 different regions of the HLA system: the HLA-A locus (HLA-A2), the HLA-DR/DQ region (HLA-DR5 and DR8 haplotypes) and the DP region (DPB1*0201). All these associations are independent of each other and not brought about by linkage disequilibrium. There are significant interactions between the associated alleles DPB1*0201 and DR5 haplotypes, DPB1*0201 and DR8 haplotypes as well as between A2 and DR5 and DR8 haplotypes, and between A2 and DPB1*0201. The association with the DR/DQ haplotypes reveal that most likely the DQA1 gene locus is primarily associated with JCA. There is a common motif which is present on all susceptible DQA1 alleles (0401, 0501, 0601) and not present on all the others (12). Taking all these information together, the following hypothesis is proposed: the observed HLA associations are a reflection of the direct involvement of the HLA genes in pathogenesis. The normal function of HLA molecules, namely the presentation of peptides of the T-cell receptor, is assumed to be a key mechanism in pathogenesis where an arthritogenic peptide is specifically bound by the DQ molecules, with binding specificity determined by the common motive on the DQA chain. It is possible that some of the arthritogenic peptides may be derived from self-histocompatibility antigens such as HLA-A2 and/or DPB1*0201.

Subtypes of HLA-DRB1*03, *08, *11, *12, *13 and *14 in early onset pauciarticular juvenile chronic arthritis (EOPA) with and without iridocyclitis.

A set of 200 patients with early onset pauciarticular juvenile chronic arthritis (EOPA-JCA) from Munich (165) and Prague (35) was investigated for the subtypes of HLA-DRB1*03, *08, *11, *12, *13 and *14. In addition, the relationship of DRB1, DQA1, DQB1 and DPB1 alleles with iridocyclitis in patients with EOPA-JCA was investigated. Subtyping for DRB1*03 was not informative, as all DR3 positive patients and all except one of the controls possessed DRB1*0301. Thus, the role of DRB1*0302 could not be assessed. The subtypes for DRB1*12, *13, and *14 did not reveal any statistically significant difference between patients and controls. In contrast, the subtype DRB1*1104 was the one most strongly associated with EOPA-JCA (chi 2 31.2, p value < 10(-6)). It appears that the subtype DRB1*1103 may also be associated with EOPA-JCA. The association of EOPA-JCA with DR8 is almost exclusively due to the subtype *0801. For the other alleles *0802, *0803, and *0804 there is no evidence for or against involvement in JCA. The analysis of iridocyclitis in EOPA-JCA revealed that DRB1*1104 is not more frequent in patients with eye disease than in patients without eye disease. The presence of DRB1*01 appears to convey some protective effect against the occurrence of iridiocylitis in EOPA-JCA, as had been previously observed by Melin-Aldana et al.