Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

Differential association of polymorphisms in the TNFalpha region with psoriatic arthritis but not psoriasis.

OBJECTIVE: To investigate the potential association of tumour necrosis factor alpha (TNFalpha) microsatellite and promoter alleles with psoriatic arthritis (PsA). METHODS: DNA from 89 white patients with PsA, 65 patients with psoriasis, and 99 healthy white controls was investigated for two TNFalpha promoter (-238 and -308) and three microsatellite polymorphisms (TNFa, c, and d). Patients had previously been studied by serology for HLA class I antigens and by sequence-specific polymerase chain reaction for DRB1* alleles. In addition, TNFalpha production of Ficoll separated peripheral blood mononuclear cells (PBMC) into culture supernatants after stimulation with lipopolysaccharide, alphaCD3 antibodies, phytohaemagglutinin, and streptococcal superantigen C was determined. RESULTS: A significant, HLA class I independent increase of the TNFa6c1d3 haplotype was found in the group with PsA but not among patients with psoriasis (32% v. 8%, pc<0.008; relative risk (RR)=5.3). In addition, patients with PsA showed a marked decrease of the TNF308A promoter allele (6% v. 18%; pc<0.008; RR=3.5) compared with healthy controls, which was independent of the increased frequency of the -238A polymorphism in this group. PBMC from patients with PsA secreted significantly less TNFalpha than cells from patients without arthritis. In particular, the TNFa6 microsatellite was associated with decreased TNFalpha production. CONCLUSION: These data indicate that allelic variations at the TNFalpha locus influence susceptibility to PsA. Decreased production of TNFalpha is at least in part genetically determined and might be related to the development of arthritis. However, the association of the TNF308G allele with the disease also points to other disease related haplotypes with still unknown susceptibility genes.

Rapid detection of the ERV-K(C4) retroviral insertion reveals further structural polymorphism of the complement C4 genes in old world primates.

The fourth component of complement (C4) is coded for by two tandem-duplicated genes located in the class III region of the MHC of humans as well as a number of primates. A C4 gene size polymorphism giving rise to two gene variants of 16 and 22.3 kb length can be attributed to a complete endogenous retroviral insertion of 6.3 kb termed ERV-K(C4) in intron 9 of the long C4 genes. We developed a simple PCR-based screening assay to detect the presence of this insertion, and tested a number of unrelated animals from old world primate species. The presence of the ERV insertion in the orangutan, rhesus macaque and green monkey as well as its absence in gorillas and chimpanzees could be confirmed. In addition, the insertion was also detected in the baboon and the cynomolgus macaque whereas it was not found in a single gibbon. Among rhesus and cynomolgus macaques one individual was identified in each species only carrying short C4 genes demonstrating further structural heterogeneity in these species. Based on these findings we propose that the primigenial retroviral integration occurred prior to the radiation of old world primate species, and that both the long and the short forms of the C4 gene have existed side by side since then.

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.

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.

Genetic polymorphism of human complement factor I (C3b inactivator) in the Chinese Han population.

The human complement factor I (IF) polymorphism has been analysed by polyacrylamide gel isoelectric focusing electrophoresis of neuraminidase-treated EDTA plasma samples followed by immunoblotting and enzymatic detection. In a population study among 121 random individuals from Chengdu, PR China, three different common phenotypes were observed. The results show that IF is polymorphic in the Chinese population. The allele frequencies were as follows: FI*A = 0.153, FI*B = 0.847. The distribution of observed phenotypes was in accordance with the Hardy-Weinberg equilibrium. In comparison to other Asian population studies, the frequency of the IF*A allele was the highest in the Chinese population studied here.

The influence of major histocompatibility complex class II genes and T-cell Vbeta 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.

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-alpha-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.

Reference typing report for complement factor B.

Factor B (BF) reference typing was carried out on the occasion of the VIIth Complement Genetics Workshop in Mainz, May 1998. Two different sets of samples were analysed by agarose electrophoresis and/or isoelectric focusing at the protein level, and by PCR-RFLP analysis at the DNA level. These results confirmed the reliability of the standard agarose electrophoresis technique for the identification of the major BF alleles as well as for the identification of cathodic and anodic variants. However, the exact alphanumeric designation of individual variants relative to the reference distance between alleles S and F1 turned out to be more difficult. Using PCR-RFLP analysis, the common alleles F and S as well as the FA and FB subtypes in 6 samples containing an F allele were all assigned correctly. However, the variants F1 and S07 were not detected by this method, as they could not be distinguished from the accompanying S allele. Therefore, a combined application of all three typing methods is recommended for a reliable identification of factor B alleles, variants and FA/FB subtypes.

Reference typing report for complement component C3.

A comparison of five C3 variant samples has been performed by conventional high-voltage gel electrophoresis in three laboratories (Palermo, Berlin and Mainz). Local designation was shown within SD = +/-0.75 mm migration distance in the Mainz laboratory. Methodological modifications by laboratories were not accounted for (cooling temperature, relative mobilities between runs). In parallel, all reference samples were also sequenced after exon-specific amplification. As a result, two variants with identical final designations and two variants with different mobilities were shown to conform at the molecular basis exhibiting an amino acid exchange that causes the corresponding change in relative mobility as compared to normal C3F and C3S.

Genetic polymorphism of the fourth component of human complement: population study and proposal for a revised nomenclature based on genomic PCR typing of Rodgers and Chido determinants.

The fourth component of human complement (C4) is coded for by two homologous genes, C4A and C4B, located in the class III region of the major histocompatibility complex (MHC). Genetic typing of C4A and B alleles is routinely carried out by high-voltage agarose gel electrophoresis. The electrophoretic C4 polymorphism can be further subdivided by the Rodgers (Rg) and Chido (Ch) blood groups, which are antigenic determinants of the C4A and B alpha-chains, respectively. We have used a recently described direct PCR typing method using sequence-specific primers (PCR-SSP) in combination with electrophoretic C4 typing as well as genomic RFLP analysis to determine the frequency of C4 allotypes, Rg/Ch subtypes and C4A-B haplotypes in a family study of the German population. As the current C4 allele designation does not provide any information about the presence or absence of Rodgers and Chido antigens, we have developed an extension to the existing C4 nomenclature. This revised allele designation combines the existing numerical allotypes defined by electrophoretic mobility with eight subtypes (01-08) based on Rg/Ch PCR genotyping results. Using this approach, most electrophoretic allotypes could be subdivided. Among the C4A allotypes, the most common allele was A*0301 (59.9%), and the most common subtype among all electrophoretic allotypes was 01 (85.1%; = Rg1,2-positive, Ch-negative). For C4B, the most common allele was B*0101 (64.3%), and the most common subtype was 01 (79.6%; = Ch1,2,3,4,5,6-positive, Rg-negative). The subtypes 03, 04, 07 and 08 of the C4A allotypes, and the subtypes 03, 07 and 08 of the C4B allotypes, were not detected in this study. The analysis of duplicated C4 alleles revealed considerable heterogeneity of their subtypes. The results demonstrate that all known C4 allotypes can now be assigned unambiguously, which facilitates the identification of MHC haplotypes relevant for transplantation and disease association studies.

A combined study of human complement C7 IEF and C7 M/N polymorphisms in the Chinese Han population.

Two polymorphisms of human complement C7 (IEF and M/N) have been studied in the Han population from Chengdu, People's Republic of China. One was determined by polyacrylamide gel isoelectric focusing (IEF) of neuraminidase-treated EDTA plasma, whereas the other was performed by a combination of two ELISAs using a C7-allospecific monoclonal antibody or polyclonal C7-specific IgG as coating antibodies. In 121 Chinese subjects IEF patterns of C7 were classified into three common and four rare phenotypes. The C7 IEF allele frequency estimates for C7*1, C7*2, C7*3, C7*4 and C7*7 were 0.831, 0.095, 0.045, 0.025 and 0.004, respectively. The C7 M/N typing in 113 Chinese individuals revealed the following allele frequencies: C7*M = 0.805, C7*N = 0.195. Allelic associations between C7 IEF alleles and the C7 M/N alleles are also discussed.

Human complement C81 (C8 A) polymorphism: detection and segregation of new variants.

In addition to the earlier detected C81(A) rare variants A1, A2 (now A3) and B1 (now B2), six new rare variants (C81 A2 new, A4, A5, A6, M1 and B1new) are described within the polymorphism of the eighth component of human complement (alpha-gamma chain subunit). Except for A3, all rare C81 A variants are only detected by isoelectric focusing, and not by SDS polyacrylamide gel electrophoresis (PAGE), in the alpha-gamma subunit. In one individual out of approximately 700 individuals studied, a reversed position of the common allele (B vs A) was observed by SDS PAGE and the isofocusing technique. The segregation of A1, A3 and A4 could be followed in putative father/child combinations.

Genetic polymorphisms of the A and B subunits of coagulation factor XIII in the Chinese population.

Coagulation factor XIIIA and XIIIB polymorphisms in a random population sample from Chengdu in southwest China (n = 121) were studied using isoelectric focusing in polyacrylamide gels followed by immunoblotting with enzyme immunoassay. The allele frequencies were as follows: FXIIIA*1 = 0.8719, FXIIIA*2 = 0.1240, FXIIIA*3 = 0.0041; FXIIIB*1 = 0.2727, FXIIIB*2 = 0.0165, FXIIIB*3 = 0.7107. The distribution of phenotypes of FXIII agrees with the Hardy-Weinberg equilibrium. Comparing these allele frequencies with those reported in other populations, it was found that the allele frequencies of FXIIIA*1 and FXIIIB*3 in the Chinese population were higher.

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.

C4 alpha-chain reference typing report.

Previously it was shown that C4A and C4B alpha-chains after separation on SDS-PAGE can provide valuable information on presence and absence, as well as the number of C4A and C4B genes expressed in an individual. All samples submitted for C4 reference typing were also subjected to C4 alpha-chain separation; the results were included in the Final C4 Reference Typing List [Complement Inflamm 1990;7:193-212]. In addition, in selected cases with assumed 'reversed antigenicity', Western blots of C4 alpha-chains with monoclonal anti-C4A and B antibodies were obtained. As a result, subtypic differences of C4B allotypes were detected by the comparison of monoclonal antibodies 1217 and 1228.

C3 reference typing report and nomenclature revision.

As the result of reference typing, two 'new' variants could be provisionally accepted (C3F045 and C3F015). The list of variants of the C3 polymorphism includes now 2 common and 29 rare variants.

C8 reference typing report and nomenclature recommendation.

Using two different typing techniques (i.e. polyacrylamide gel isoelectric focusing (PAGIF) with Western blot and SDS-polyacrylamide gel electrophoresis of precipitated C8 under nonreducing conditions with Western blot), the following observations were made during the reference typing for C81 (C8A). The Japanese variant A1J is probably identical with A1Cauc, whereas B1J is definitely different from B1Cauc and could therefore provisionally be named HB3'. Variant 'A2' from Japan is focused in an intermediate position, but different from M1 and could be named 'M2'. Both variants possess normal A subunits. B2 from Japan is clearly different from B1Cauc and should retain its designation. In PAGIF, its subunit has a position more anodal than A. Summarizing these results, more information is needed before a final nomenclature can be proposed.

C4 reference typing report.

Human C4 is most polymorphic at the protein level, distinction between allotypes of the C4A and C4B proteins resting on electrophoretic migration patterns and difference in hemolytic activity. The aim of the C4 reference typing has been the definition of reference variants, the assignment of rare variants, and the investigation of duplicated, deleted, or non-expressed and hybrid genes. Samples from 136 individuals, predominantly with known segregation, from 16 laboratories were investigated by standard electrophoretic techniques, for their relative hemolytic activity, reactivity with monoclonal antibodies and Rg/Ch reagents, alpha-, and beta-chain types, relative electrophoretic migration distance, as well as the C4/21-OH-TaqI RFLPs. The results were evaluated in three groups; they consisted in the definition of the eight most common C4 alleles, and the ten Rg/Ch standard phenotypes in group I. In group II twelve C4A and fourteen C4B duplications among 96 complotypes, as well as eighteen deleted/non-expressed C4A and twenty-two C4B alleles, and hybrid alleles were seen by correlation of lytic activity, electrophoretic mobility, and monoclonal and/or Rg/Ch reactivity. Group III consisted of the newly defined allotypes A 8, A 7, A 58, A 55, A 45, B 45, B 35, and B 22, furthermore of alleles subdividing the A 1/A 91, and the B 13/B 12/B 11 regions. The reference typing has allowed reclassification of the majority of described C4 allotypes and resulted in a revision of the C4 nomenclature.