Beate Stradmann-Bellinghausen

HLA DRw8 and complement C4 deficiency as risk factors in primary biliary cirrhosis.

HLA class I, II, and III alleles were investigated in 25 consecutive unrelated German patients with primary biliary cirrhosis and in two families with two primary biliary cirrhosis patients in each. In primary biliary cirrhosis patients, HLA class I antigens did not differ significantly from in health controls. For HLA class II antigens, a highly significant increase of HLA DRw8 was found in patients with primary biliary cirrhosis compared with controls. Thirty-six percent vs. 3.6% were DRw8 positive [relative risk = 15.28; P (corrected) = 0.00013]. The genetic typing of HLA class III alleles revealed an increased incidence for C4AQ0 alleles [72% vs. 34.5%, relative risk = 4.89: P (corrected) = 0.0056]. A highly significant proportion of primary biliary cirrhosis patients carrying both DRw8 and C4A-Q0 alleles (relative risk = 183.75; P = 9.7 x 10(-7)) were found. In one family, a mother and her daughter had primary biliary cirrhosis, both sharing the major histocompatibility complex haplotype HLA-A1, -B8, -DR3, -C4AQ0B1. In the other family, two sisters with primary biliary cirrhosis shared the major histocompatibility complex haplotype HLA-A24, -B8, -DRw8, -C4A4B2. These studies contribute to the further elucidation of the immunogenetic background of primary biliary cirrhosis.

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier’s algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

Forensic validation of the SNPforID 52-plex assay

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations. The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) reactions which are detected with capillary electrophoresis. In order to validate this assay, a variety of DNA extracts were chosen to represent problems such as low copy number and degradation that are commonly seen in forensic casework. A total of 40 extracts were used in the study, each of which was sent to two of the five participating laboratories for typing in duplicate or triplicate. Laboratories were instructed to carry out their analyses as if they were dealing with normal casework samples. Results were reported back to the coordinating laboratory and compared with those obtained from traditional STR typing of the same extracts using Powerplex 16 (Promega). These results indicate that, although the ability to successfully type good quality, low copy number extracts is lower, the 52-plex SNP assay performed better than STR typing on degraded samples, and also on samples that were both degraded and of limited quantity, suggesting that SNP analysis can provide advantages over STR analysis in forensically relevant circumstances. However, there were also additional problems arising from contamination and primer quality issues and these are discussed.

C4A deficiency and nonresponse to hepatitis B vaccination

BACKGROUND/AIMS Hepatitis B vaccination failure has been linked to the presence of certain human leukocyte antigen class II alleles. However, the functional background of these associations has remained unclear. Complement component C 4 is encoded within the major histocompatibility complex and is essential for classical pathway activation. METHODS Healthy individuals (n=4269) were vaccinated in a prospective trial with Engerix B. Nonresponse was classified as anti-HBs<10 U/l after the last vaccination. Seventy-three nonresponders (NR) (1.7%) were identified. For comparison 53 responders (R) (anti-HBs>10 IU/l) were drawn randomly from the same cohort. C4 allotyping was carried out by high-voltage agarose gel electrophoresis and C4alpha-chain typing using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C4 gene deletions (C4Del) were studied by Southern blot. RESULTS C4AQ0 alleles were observed in 45/73 (62%) NR compared to 17/53 (32%) R (P=0.001). C4ADel was observed in 24/73 (33%) NR and in 6/52 (12%) R (P=0.006). C4AQO alleles were present in 21/49 (43%) NR without C4Del compared to 10/46 (22%) in R without C4Del (P=0.031). In a logistic regression with DRB1*0301, DRB1*07, DRB1*1301 and C4AQ0 all except for DRB1*0301 showed a significant association. CONCLUSIONS C4AQ0 shows a DRB1*0301 independent association with vaccine failure. C4AQ0 alleles probably contribute to inefficient complement activation and failure of B cells to secrete anti-HBs.

IL10.G Microsatellites Mark Promoter Haplotypes Associated With Protection Against the Development of Reactive Arthritis in Finnish Patients

OBJECTIVE To investigate the association of microsatellites and single-nucleotide promoter polymorphisms (SNPs) in the gene for the cytokine interleukin-10 (IL-10) with susceptibility to and outcome of reactive arthritis (ReA). METHODS From genomic DNA, IL-10 microsatellites G and R and IL-10 promoter polymorphisms at positions -1087 and -524 were typed by polymerase chain reaction, automated fragment length analysis, and restriction fragment digestion in 85 Finnish patients with ReA and 62 HLA-B27-positive Finnish controls. ReA patients had been followed up for 20 years. Genotypes and haplotypes of IL-10 were correlated with distinct features of the disease course, such as triggering agent, chronic arthritis, development of ankylosing spondylitis, and other chronic features. RESULTS There was a significant decrease in the promoter alleles G12 (allele frequency 0.206 versus 0.033; corrected P < 0.001, odds ratio 0.14) and G10 (0.183 versus 0.092; P < 0.05, odds ratio 0.44) in the ReA group compared with the HLA-B27-positive controls. Chronic arthritis developed significantly more frequently in the B27-positive subjects than in the B27-negative subjects (P < 0.05) as well as in patients with [corrected] the IL10.G8 allele. No associations were observed for either SNP or for the IL10.R microsatellite polymorphism. CONCLUSION IL10.G12 and G10 microsatellite alleles show a strong protective effect against the development of ReA in Finnish subjects. Since these polymorphic markers themselves do not have direct functional implications, they most likely mark promoter haplotypes with distinct functional properties, suggesting that differential production of IL-10 is an important susceptibility factor for the development of ReA.

The Influence of Major Histocompatibility Complex Class II Genes and T-Cell Vβ Repertoire on Response to Immunization with HBsAg

Nonresponsiveness to HBsAg vaccination is observed in 5-10% of vaccine recipients and is possibly caused by a defect in the T helper cell compartment. The immune response to HBsAg is influenced by genes of the major histocompatibility complex. We have investigated MHC class I and class II antigens in 53 adult responders and 73 nonresponders. Results obtained in this first study were tested in a second study with 56 responders and 62 nonresponders from an infant vaccination trial. In addition, the peripheral Vbeta-chain T-cell receptor repertoire was investigated using monoclonal antibodies and flow-cytometry in 26 adult responders and 38 nonresponders. As previously reported, nonresponsiveness to HBsAg vaccination was associated with DRB1*3 and DRB1*7. In addition, DRB1*13 was significantly increased among vaccine responders (35.2% vs 5.4%;p < 0.0001) suggesting an immune response promoting effect for this allele whereas the closely related allele DRB1*14 was associated with nonresponse in the infant study. There was no evidence for a hole in the T cell receptor Vbeta repertoire. In conclusion, in agreement with results obtained in mice there appears to be a hierarchy of DRB1* genes in the HBsAg immune response. The possible differential association of DRB1*13 and DRB1*14 may allow the identification of differences between responsiveness and nonresponsiveness to a few amino acid differences in the beta1-domain of the class II heterodimer.

Sequence polymorphism of mitochondrial DNA control region in Japanese

Sequence polymorphisms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II, from 100 unrelated Japanese were determined by PCR amplification and direct sequencing. Sequences of 404 nucleotides for hypervariable region I and 379 nucleotides for region II were obtained. Variable sites (85 and 45) were revealed in region I and region II, respectively, as compared to the reference sequence, and a total of 96 different genetic patterns from both regions I and II were determined. A point mutation heteroplasmy was observed at the ratio of approximately 50:50 from one individual at the sequence position 151 showing a nucleotide transition from C to T. The probability of identity was estimated as 2.3% for region I, 3.9% for region II, and 1.1% combined for both regions. These results suggest that sequence polymorphism of mtDNA control region would be very useful in forensic practice as a marker for individual identification.

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories. Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male). All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data. As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland. Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.

Reference Typing Report for Complement Component C4

During the 7th Complement Genetics Workshop, Mainz, Germany, May 1998, a complement component C4 typing exercise took place with the aim of applying present technologies to the definition of reference C4 alleles/phenotypes and the recognition of nonexpressed (Q0) C4 alleles within expressed haplotypes. Eleven samples were submitted from 3 laboratories and tested by 14 participating laboratories with basic protein-typing technologies; in addition, each laboratory contributed data from local expertise. The samples were introduced to the reference typing for one or more characteristic allotype or for partial or total nonexpression of one isotype. The blinded samples were centrally evaluated and the results discussed among the participants at a plenum meeting. From the results, the samples could be classified into a group of common, easy to diagnose pheno-/allotypes, less common but still unanimously recognised variants, and a third group with difficult pheno-/allotypes. Within the latter group, the allotypes were either new (C4A ‘92’; C4B ‘93’) and/or showed partial or total reversed antigenicity and unusual Rodgers/Chido (Rg/Ch) PCR subtypes (C4A ‘92’; C4A 12; C4B ‘35’; C4B ‘13’). Semiquantitative C4-α-chain estimates of relative isotype levels correlated well with the number of alleles seen at each locus by agarose gel electrophoresis, and were superior to other isotype quantitation methods. From the evaluation of the reference typing it was concluded that the recognition of rare, aberrant or hybrid C4 alleles with partial or total reversed Rg/Ch antigenicity or monoclonal reactivity is still difficult in most instances; besides isotype-dependent lysis, relative migration values, immunoblots with Rg- and Ch-specific monoclonal antibodies, Rg/Ch PCR typing, side-by-side comparison with already described allotypes will ultimately be required. The recognition of nonexpressed alleles within C4A and C4B expressed phenotypes remains the major obstacle in C4 genetic typing. Finally, a conclusive interpretation of DNA typing results will be achieved only in the context of complete allotyping results at the protein level, and at present cannot replace conventional protein allotyping.

Development of a quadruplex PCR system for the genetic analysis of X-chromosomal STR loci

Short tandem repeat systems on the X chromosome are the natural counterpart to the well-established Y-chromosomal STR loci. The X-linked systems are inherited as a single haplotype only in males, whereas in females, the X chromosomes recombine and exhibit the same characteristics as the autosomes. Nevertheless, X-linked systems may provide a useful tool in paternity cases with female offspring, in particular when the alleged father is not available for testing, or in forensic identification cases based on the comparison with firstor second-degree relatives. Only a small number of STR loci have been described on the X chromosome, and a number of these are not highly informative. Therefore, we have investigated the four X-chromosomal STR loci: DXS101, DXS8377, HPRTB and STRX-1 [1–5], to develop a rapid multiplex PCR typing system suitable for fluorescent detection and to perform population genetic studies among Germans. Whereas HPRTB and STRX-1 contain tetrameric repeats, DXS101 and DXS8377 have trimeric repeats. All four loci are highly polymorphic exhibiting up to 18 common alleles at each locus (Table 1).