No abstract

In order to obtain reliable information on HLA types, DNA typing with sequence-specific oligonucleotide/primer (SSO/SSP) typing sets or sequencing-based typing (SBT) is increasingly performed. The quality of the evaluation depends on the presence of a complete listing of all typed alleles as well as on the ability of detecting all corresponding alleles/allele pairs. We have developed the concept of virtual DNA analysis (VDA), which is able to combine all types of SSO/SSP/SBT results and evaluate this typing in combination according to the latest published allele sequence lists. The concept is based on the target DNA recognised by the respective typing techniques. All SSO/SSP or SBT results are transformed to a virtual sample DNA, which subsequently is analysed. Evaluation of generic or allele-specific DNA typing or the combination of both is supported. Due to this flexible approach, all kinds of SSO/SSP sets, as far as the respective SSO/SSP sequences are available, can be entered and evaluated immediately. The combination of collected data of different typing sets and procedures leads to the highest possible typing resolution. If more than one possible allele combination persists, the program reduces the result to the most specific common denominator in a stepwise manner. VDA offers the possibility of re-evaluation of former SSO/SSP/SBT results, alone or in combination. No solutions are omitted. This might be a first step towards standardisation of evaluating DNA-based HLA typing results or transfer of the respective typing data for later evaluation.

No abstract

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.

No abstract

Wegener's granulomatosis (WG) and microscopic polyangiitis are systemic autoimmune diseases characterized by the presence of ANCA in the sera of patients. Little is known about the aetiologic factors and genetic predisposition as well as the pathogenesis of these disease entities. A slightly decreased representation of HLA-DRB1*13 and HLA-DQB1*0603 individuals was observed in our cohort of ANCA-associated systemic vasculitis (AASV) patients compared with controls. In addition, HLA-DRB1*04 individuals were over-represented in a subgroup of patients with WG in end-stage renal disease as a result of renal vasculitis. In order to identify other genes relevant for these diseases, we investigated highly polymorphic markers in the vicinity of several immunorelevant genes, i.e. tumour necrosis factor (TNF)alpha, IL-2, IL-5 receptor alpha (IL-5RA), in a group of 102 patients with AASV and compared the representation with controls. Furthermore, functional polymorphisms were directly analysed in the promotor region of TNFalpha as well as in the coding region of the FcgammaIIRA genes. Polymorphisms of the TNFalpha promotor (TNF-308) as well as in the FcgammaIIRA gene were excluded as risk factors for the disease in our cohort. No major phenotype distribution differences were observed between patients and controls for the IL-2 and IL-5RA microsatellites. Most importantly, several haplotypes on chromosome 6p appeared strongly associated with proteinase 3 (PR3)-ANCA+ AASV. Thus, as in other autoimmune diseases, different predisposing factors play differential aetiopathogenic roles in various groups of AASV patients.

DNA profiles (immunoprints) were generated for 120 patients suffering from early onset pauciarticular chronic arthritis (EOPA-JCA) and > 500 healthy controls utilizing highly polymorphic microsatellites in the vicinity of immunorelevant genes. Six T cell receptor (TCR) markers for the CD3D, TCRDVAJ, TEA, TCRBV6S1, BV6S3, BV6S7 and BV13S2 genes were analysed. Furthermore markers for the cell surface molecule CD40L, for cytokine genes (IL-1A, IL-2, IFN-alpha, FGF-alpha, TNF-alpha), the chromosomal region of the IRF2 and the cytokine receptor gene IL5RA were studied as well as two polymorphisms within the promotor region of the TNF-alpha gene. Coding region polymorphisms were evidenced indirectly by repeat length variation or they were predicted from the microsatellite distribution profiles and then confirmed by direct sequence analysis. Statistical evaluations were performed with respect to known predispositions, predominance of females (> 80%) and HLA-DR and -DQ haplotypes. Cell surface molecules (TCR, CD40L, IL5RA) as well as almost all cytokines (IL-1A, IFN alpha, FGFA, IRF2 region) were excluded as predisposing in our JCA panel. The TNF-alpha microsatellite alleles (GT)10-12 contribute considerably to manifestation of the disease, in HLA-DRB1*11(12) individuals (RR = 12.8). The TNF-alpha allele is not found in linkage disequilibrium with HLA-DRB1*11(12) and may be present on either chromosome 6. Thus, a novel susceptibility factor probably within the TNFA/TNFB gene region has been identified via linkage with the TNF-alpha microsatellite allele. Apparently complex compositions of the genetic background rather than single genes provide the precondition for manifestation of the autoimmune disease EOPA-JCA. Immunoprinting unravels the variability of the immunological genome via the semi-directed microsatellite approach efficiently.

We have sequenced exons 2, 3 and 4 of MIC‐A in 23 homozygous cell lines, 22 families and 54 unrelated individuals. This has led to the definition of seven polymorphic positions in exon 2, 13 in exon 3 and 12 in exon 4, yielding a total of 33 different MIC‐A allelic specificities, of which 16 have not been described before. The newly defined sequences and those of the alleles defined before were entered into a database of the SCORE program (Helmberg et al., 1998, Tissue Antigens, 51, 587) for comprehensive genotyping analysis. In the tested sample, only one genotype present in two individuals gave rise to an ambiguous genotype. If all possible combinations of the 33 alleles are considered, 10 of 636 combinations are ambiguous. The MIC‐A exon 2, 3 and 4 polymorphism is characterized by diallelic single base exchanges and by a considerable degree of exon shuffling. The majority of heterozygote positions identified are non‐synonymous, i.e. five of seven in exon 2, 13 of 13 in exon 3 and eight of 12 in exon 4, suggesting an important function for the MIC‐A polymorphism.