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52 symptomatic (SPB) and 64 asymptomatic (APB) pigeon breeders were investigated for the HLA-A, -B, -C, -DR; C2, C3, C4 and Bf systems, and C3, C4 and factor B serum concentrations. HLA-DR3 and Bf S frequencies were significantly higher in the SPB than the APB group. Mean factor B concentrations were lower in the SPB than in the APB group. A positive two-way association between HLA-DR3 and the disease was found. It is concluded that a gene or genes responsible for type III and IV allergic reactions leading to the disease is associated with HLA-DR3. The increase of Bf S allotype and low mean factor B concentrations, however, can be explained by the strong linkage disequilibrium between BfS and HLA-DR3.

Two series of 88 and 53 patients with Down's syndrome and 91 other mentally retarded patients were investigated for various blood, serum and enzyme groups. No association of trisomy 21 and the following systems and factors was found: ABO, Rh(D), MNSs, K, P, Fy(a, b), Jk(a, b), Gm, (1,2), InV(1), Ag (x), Lp(a), Tf, C3, acP, PGM1 and GPT. None of these markers was associated with age or the presence or absence of the Au/SH antigen in Down's syndrome or other mentally handicapped patients. Gm(+1) individuals were more frequent among Down and other mentally retarded patients than in normal controls. A decrease of Hp2 individuals with age was found in the first, but not in the second group of Down patients. A possible association of Au/SH positive and tic 1-1 individuals was found in the first. but not confirmed in the second series of patients with Down's syndrome. The control group of other mentally retarded patients exhibited a helerogeneity according to sex and age in the distribution of Gc phenotypes.

SUMMARY It is described that HL-A inhibiting substances can be detected in dried human blood stains. An inhibition test is used to evaluate stability and specificity of soluble HL-A antigens in one- and two-stage methods. With few exceptions, the occurrence of inhibition and non-inhibition of stains from different subjects in respect to given HL-A antisera is in agreement with specificities present on the donor's cells. Some‘weak inhibitors’were observed. Extra-reactions with given antisera can be explained by cross-inhibition. One- and two-stage methods are evaluated with regard to the inhibition test for human blood stain HL-A typing.

1. IntroductionEngraftment of donor stem cells after allogeneic bone marrow transplantation can begenetically monitored by PCR typing of DNA polymorphisms [1]. Successful engraftmentwith complete chimerism and presence of the donor’s genotype in the bone marrow has tobe demonstrated, and the presence of the patient’s alleles has to be excluded. Detection ofthe patient’s alleles provides evidence for an incomplete chimerism or for a relapse ofmalignant disease. STRs have been used successfully for this type of genetic monitoring[2]. For the present study, we have developed an approach to quantify the ratio of donorchimerism using mock mixture experiments. The usefulness of our approach is demon-strated in typical cases where the donor chimerism could be monitored over periods ofmore than 1 year after transplantation (Tx).2. MethodsThe degree of donor–recipient chimerism was monitored by a semi-quantitativeanalysis of the mixed STR genotypes. DNA was extracted from bone marrow samplesas well as from FACS-separated mononuclear cells, CD3+ T cells and CD3 mono-nuclear cells. Furthermore, buccal swabs were collected from donor and recipient toestablish their genotypes prior to transplantation. The DNA was subjected to PCR typing

1. IntroductionThe study of Y chromosomal haplotypes and their relationship to human evolution andvariation is increasing rapidly in the fields of anthropology and forensic genetics.Although autosomal STRs are commonly used and very informative for paternity testingand forensic identification, the use of the nonrecombining portion of the Y chromosome isimportant and provides additional data in cases when the offspring is a male or for mixedmale/female crime stains. For this purpose, in the past 2 years, more and more attentionhas been paid to the examination of diallelic polymorphisms (SNPs) on the Y chromosome[1].Here, we describe an approach for the typing of 10 Y-chromosomal diallelic poly-morphisms (SNPs) using single base extension technology (minisequencing).The frequencies and haplotype relationships of diallelic polymorphisms (Table 1) in asample of 95 individuals of Caucasian origin were examined. In addition to the SNPs, wealso tested the Y-chromosomal diallelic Alu repeat insertion DYS287 (YAP) [2] as well as

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).

Interleukin-10 (IL-10) has a critical role in the regulation of immune responses. The relative contribution of genetic and environmental factors to IL-10 production is under debate. We performed a twin study in 246 monozygotic and dizygotic twins to assess the heritability of IL-10 production after LPS stimulation in whole blood. In addition, the influence of promoter single nucleotide polymorphisms (-1082, -819 and -592) on transcriptional activity and their binding to nuclear factors was studied in luciferase reporter gene and electrophoretic mobility shift assays. IL-10 production showed a genetic determination with a heritability of 0.5. Decreasing body mass index (BMI), smoking and female gender lead to decreased IL-10 production. In monocytes, the -1082A allele showed higher binding affinity to the transcription factor PU.1 resulting in decreased transcriptional activity of -1082A promoter haplotypes. Genetic determination of IL-10 secretion is probably lower than that previously reported. Fifty percent of the observed variability explained by genetic factors. Female individuals produce less IL-10 than male subjects. Environmental factors like smoking and decreasing BMI exert suppressing effects on IL-10 production. Although the -1082A allele shows higher binding affinity to the PU.1 transcription factor and lower transcriptional activity, this polymorphism probably explains only a small fraction of the observed heritability.

Summary C9 is the last of the human complement components creating the membrane attack complex. The single chain serum protein is encoded by a gene located on chromosome 5p13 that is composed of 11 exons. With the aid of inverse PCR, the hitherto unknown regions flanking exon 1 and the 3′ part of exon 11 (3′UTR) have been sequenced. A computer-based analysis of the 300-bp region located just upstream of the AUG start codon showed homologies to known DNA modules which affect the transcriptional regulation of certain genes. The most striking of these is a sequence that may substitute the missing TATA box in initiating C9 transcription. In the 3′UTR, three successive polyadenylation signals were found. Although the C9 protein is invariant, four different single nucleotide polymorphisms (SNPs) have been observed at the DNA level by exon-specific PCR and direct sequencing. None of them changes the amino acid composition of the mature protein. Due to a C T transition in exon 1 at cDNA position 17, the fifth amino acid of the leader peptide may be either an arginine or a tryptophane. Using either PCR/RFLP analysis (exons 1 and 11) or allele-specific PCR (intron 1 and exon 4), each polymorphism can be characterized without sequencing. All of the exon 1, intron 1 and exon 11 variants could be detected in small population samples of European, Thai or South American Indian origin. In contrast, the exon 4 C variant was observed only once in a European. The first three SNPs can be combined to designate eight different ‘C9 alleles’. Of these, six have actually be found. These data provide strong evidence that several mutation and recombination events occurred in the course of C9 gene evolution.

Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

The reconstruction of accident and crime scenes demands the full attention of the forensic working physician. Description by words is often difficult and liable to be misunderstood. Reconstruction in the original places of events are expensive and in some cases impossible. Computer graphics and animations give the possibility to construct the original course of events. Poser4 is a software package to perform these reconstructions in an easy and vivid way. We investigated the possibilities of reconstructing an accident with this software.

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.

Variants of the mannose-binding lectin (MBL) have been shown to be associated with low serum concentrations of the protein and to predispose to bacterial, fungal and viral infections. A recent small study on 33 Caucasian patients had suggested that a mutation at codon 52 of the MBL gene is associated with chronic hepatitis B virus (HBV) infection. Exon 1 of the MBL gene was amplified by PCR in 61 patients with chronic HBV infection, 28 patients with acute infection and in 60 controls. MBL variants were detected by subsequent restriction enzyme digestion and agarose gel electrophoresis. The occurrence of the codon 52 mutation in patients with chronic HBV infection did not differ significantly from that in controls or patients with acute infection (9 vs. 7%), nor were there any significant differences for the codon 54 mutation. The frequency of MBL variants at codon 52 and 54 is not increased in patients with chronic HBV infection. Thus, the previously reported association of MBL deficiency with chronic HBV infection in adults could not be confirmed.

SUMMARY 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 exisiting 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 computer simulation program (Forensic X-ray Simulation and Identification System, FoXSIS) is presented. FoXSIS calculates conventional X-ray summation images using any scanning parameters from three-dimensional CT data records. All those parameters decisive for X-ray morphology are freely selectable for realistic simulations: focus-object distance, object-film distance, centring of the X-ray beam, the location of the object in the path of rays, brightness and contrast, as well as parallel and central projection. In addition, distance and angle measurements, as well as enlargements of details are possible. The program may be expected to help in the identification of unknown corpses now, and even more in the future on account of the increased use of clinical computed tomographies.

Dental pulp tissue could be obtained in most cases from materials obtained under experimental conditions and from forensic casework (air accidents, burned and putrefied bodies). Teeth extracted during dental treatment (n = 30) were stored for 6 weeks and 4 years at room temperature. In addition teeth (n = 10) extracted from jaw fragments that had been stored for 15 years at room temperature, and teeth extracted post mortem from actual identification cases (n = 8) were investigated. Following extraction from dental pulp tissue the DNA concentration was measured by fluorometry. The amount of DNA obtained from the dental pulp tissue of a single tooth varied from 6 micrograms to 50 micrograms DNA. In most cases high molecular weight DNA was still present although the major portion consisted of degraded DNA. Genomic dot blot hybridization for sex determination using the biotinylated repetitive DNA probe pHY 2.1 was performed and sex was correctly classified in all cases using 50-100 ng target DNA. PCR typing of the HLA-DQ alpha and ApoB 3' VNTR systems from dental pulp tissue DNA was in agreement with the results obtained from blood, bloodstains, or lung tissue. In addition, Southern blot analysis of selected samples using the single locus VNTR probe pYNH24 was successfully performed. In all cases the DNA recovered from dental pulp was unsuitable for multilocus probe analysis.

A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library. A repetitive sequence of 27 bp length was identified which is contained in the probe approx. 40 times. The MZ 1.3 repeat shows variable homology of 53–73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome. Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI PvuII , and Rsa I. An average of 18 polymorphic fragments was observed using Hinf I as enzyme. The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 ± 7.2%. An example for the application of MZ 1.3 to paternity testing in an incest case is given. The probe can be used with radioactive or non-radioactive detection systems. An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.

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.

The two genes for the C4A and C4B isotypes of the fourth component of human complement are located in the MHC class III region. Previous studies have demonstrated the unusual expression of C4 genes in the form of aberrant or duplicated haplotypes. Null alleles of C4A or C4B (AQ0 or BQ0) have been defined by the absence of gene products and occur at frequencies of 0.1-0.3. However, only some C4 null alleles are due to gene deletions, the remainder were thought to be nonexpressed genes. We have analyzed the C4 gene structure of 26 individuals lacking either C4A or C4B protein. The DNA of individuals with apparently nonexpressed C4 genes was tested for the presence of C4A- and C4B-specific sequences using restriction fragment analysis and isotype-specific oligonucleotide hybridization of DNA amplified by polymerase chain reaction. All nondeleted AQ0 allels had C4A-specific sequences and may thus be described as pseudogenes, whereas the nondeleted BQ0 alleles had C4A-instead of C4B-specific sequences. Gene conversion is the probable mechanism by which a C4A gene is found at the second C4 locus normally occupied by C4B genes.

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.

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.

OBJECTIVE—To investigate the potential role of the HLA-linked LMP2 (low molecular weight protein) gene polymorphisms in conjunction with DR4 and DR7 on extraspinal disease manifestations in HLA-B27 positive patients with spondyloarthropathy. METHODS—172 patients with spondyloarthropathy, 46 healthy, HLA-B27 positive blood donors, and 99 unrelated controls were typed for HLA-class I and II antigens. LMP2 alleles were determined by polymerase chain reaction and subsequent restriction enzyme digestion. RESULTS—There were statistically non-significant increases of DR4 and DR7 in spondyloarthropathy subjects. However these differences did not relate to specific extraspinal manifestations. There were no significant differences in the LMP2 genotype distribution in the disease groups. All differences in LMP2 genotype frequencies disappeared when correcting for DR4, which was in linkage disequilibrium with the LMP2B allele. CONCLUSIONS—There is no independent association between LMP2 genotypes and the occurrence of uveitis or peripheral arthritis in HLA-B27 positive subjects with spondyloarthropathy. DR4 and DR7 do not contribute to these disease manifestations.

HLA-class II promoters contain a set of conserved regulatory regions necessary for constitutive and induced gene expression. For the HLA-DQB as well as for the DRB1 promoter sequence, polymorphisms with influence on gene expression have been reported. In contrast to these data we could show that there is very limited allele-specific polymorphism among the HLA-DRB1 promoter alleles. In a long range PCR we amplified a DNA sequence containing the promoter and the second exon of the DRB1 gene in one fragment. Nested PCR products of this PCR fragment for the promoter and for the second exon were analysed by DNA sequencing to allow the linkage of a promoter to its DR allele. Most investigated DRB1 alleles exhibited the same promoter consensus sequence except for two point mutations. An A to T transversion (position -70 bp) was closely associated with DRB1*08, whereas a C-deletion (position -30 bp) was most commonly observed together with DRB1*10. Both polymorphisms did not influence promoter activity in luciferase reporter gene assays.

Although venous air embolism is a known complication in medical practice in general, only a single case of upper gastrointestinal endoscopy complicated by venous air embolism with consecutive acute cardiovascular failure has so far been described in literature. Here we show that gastroscopy may be accompanied by massive, i.e. fatal venous air embolism. If a vessel in the gastrointestinal tract is exposed but does not collapse (in the case of a gastric ulcer, for example) air insufflated under pressure by the gastroscope may lead to a fatal air embolism. Our tests using a commercial gastroscope revealed that an overpressure of up to 43 kPa (kiloPascals) is reached without the rinsing function while an overpressure of up to 45 kPa is measured if the rinsing function is operated simultaneously. The maximum flow rates without resistance were 100 ml/min for rinsing liquid (purified water) and 2000 ml/min for air. Our results suggest that air insufflation by the gastroscope may result in a critical air embolism within very few seconds on condition that a connection with the vascular system exists. However, this complication is extremely rarely encountered. We propose that CO2 should be administered in place of air or alternatively the maximum pressure should be considerably reduced to avoid a fatal outcome in routinely performed gastroscopical examinations.

A 32-year-old patient died of a cerebellar haemorrhage and the blood coagulation analysis before death suggested defective synthesis of vitamin K-dependent clotting factors due to vitamin K deficiency. The post-mortem toxicological examination of different tissues revealed phenprocoumon poisoning as the cause of death. The differential diagnosis of vitamin K deficiency and the toxicology of hydroxycoumarins are discussed.

Complement component 3 (C3) is the central molecule of the complement system. It displays a number of polymorphic variants with, as yet, unclear functional consequences. We have investigated a number of rare C3 variants by PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) analysis and could identify the molecular basis of a C3*S025 variant. The decreased electrophoretic mobility of this protein is caused by the exchange of a neutral serine residue to an arginine residue (positively charged). This exchange is unlikely to have functional consequences as it maps to the C-terminus of the alpha-chain. C3 variants appear to have originated from various independent mutations as we could not detect this mutation in different allotypes.

We have applied the technique of PCR-SSCP (polymerase chain reaction-single stranded conformation polymorphism) to characterise the molecular basis of cholinesterase deficiency and variants in a Jordanian family. PCR-SSCP proved to be a quick and sensitive method of screening cholinesterase variants in a clinical setting. An AG insertion at position 351 was found to cause a silent allele, for which the parents were heterozygous and three children homozygous. In addition, the father and two sons were heterozygous for an A to G transition at position 209, known to cause the dibucaine resistant variant. No linkage to the K variant was found, which has been reported previously in white populations. These findings suggest considerable homogeneity in the molecular basis of CHE variants between different ethnic groups.

The fourth component of the human complement system (C4) is coded for by two genes, C4A and C4B, located within the MHC. Null alleles of C4 (C4Q0) are defined by the absence of C4 protein in plasma. These null alleles are due either to large gene deletions or to nonexpression of the respective genes. In a previous study, evidence was obtained for nonexpressed defective genes at the C4A locus, and for gene conversion at the C4B locus. To further characterize the molecular basis of these non-expressed C4A genes, we selected nine pairs of PCR primers from flanking genomic intron sequences to amplify all 41 exons from individuals with a defective C4A gene. The amplified products were subjected to single-stranded conformation polymorphism (SSCP) analysis to detect possible mutations. PCR products exhibiting a variation in the SSCP pattern were sequenced directly. In 10 of 12 individuals studied, we detected a 2-bp insertion in exon 29 leading to nonexpression due to the creation of a termination codon, which was observed in linkage to the haplotype HLA-B60-DR6 in seven cases. In one of the other two individuals without this mutation, evidence was obtained for gene conversion to the C4B isotype. The genetic basis of C4A nonexpression in the second individual is not yet known and will be subject to further analysis.

In contrast to the extensive knowledge about protein biochemistry of keratinization, little is known about the fate of nuleic acids during these processes (1). It has been suggested that both DNA and RNA completely degenerate in the initial phase of keratinization. However, from electron microscopical studies it is known that nuclear remnants are present (1,3). From these findings and own observations one might expect that nucleic acids should still be available.

Recent advances in molecular biology methods have significantly increased the ability to detect genetic variation at the genomic level for forensic purposes. However, the quality requirements for blood, fresh or frozen tissue as a source of DNA are a practical limitation for typing the victim in order to conduct investigations on unsolved cases. Since paraffin embedded specimens are easily obtainable the ability to study this material would be of great value in current forensic practice.

Since minisatellite DNA probes are used for the detection of hypervariable loci in eucaryotic genomes [1] the application of so called DNA fingerprints and DNA technology itself in paternity testing and forensic casework is critically discussed ([3]; Brinkmann et al., this volume). A particular problem is the possibility of obtaining partially digested genomic DNA in casework after treatment with restriction enzymes leading to inconclusive or even false results. This is even more important when multilocus DNA probes are used, since the total number of fragments in a given person is not known in advance. But also with single locus probes, where only two allelic fragments are usually detected, a fragment shift of additional bands may be produced by partially digested DNA.

Soon after the discovery of the ABO blood groups by Landsteiner their forensic application was suggested by Max Richter at the 74. Versammlung Deutscher Naturforscher und Arzte at Karlsbad (Karlovy Vary) in 1902 [1], Landsteiner’s Nobel lecture “Uber individuelle Unterschiede des menschlichen Blutes” in 1930 [2] provided a landmark on the path towards the discovery of so-called hypervariable minisatellite regions of human DNA by Alec Jeffreys in 1985 [3]. Since the individual patterns of any human being seemed to be unique it is well understood that this achievement was immediately welcomed with great enthusiasm by the scientific community and the public opinion. Soon thereafter, however, strong criticism was raised by claiming that such a high degree of individualization may violate human personality rights [4, 5]. In addition, premature and unprofessional application of DNA fingerprinting in Court cases in USA threatened to invalidate the whole procedure [6, 7]. However, the critics have also been criticized [8–10].

A dog was suspected of having caused a traffic accident. Three hair fragments were recovered from the damaged car and subjected to DNA sequence analysis of the canine mitochondrial D-loop control region. The results were compared to saliva and hair samples from the alleged dog, as well as to control hair samples from four unrelated dogs of different breeds. Two sequence types exhibiting five nucleotide differences in a 377 bp fragment were identified among the four controls. Whereas the evidence hair fragment was identical to the type 1 control sequence, the alleged dog shared the type 2 control sequence except for one position. Thus the dog could be excluded as the origin of the hair fragment. As canine mtDNA appears to exhibit only limited polymorphism, mitochondrial D-loop sequence comparison is currently only suitable for exclusions.

DNA extracted from 33 postmortem muscle specimens was analyzed using MZ 1.3, a hypervariable minisatellite probe, as well as locus-specific minisatellite probes (g3, MS1 and MS43). After storage at -25 degrees C for 10 months, DNA from all the samples was partially (approximately 21% of total DNA) degraded even when autopsy was performed 1 day postmortem. However, more than 90% of DNA samples up to at least 3 days postmortem were suitable to obtain good restriction fragment length polymorphism (RFLP) patterns. When small strips of specimen were stored for 8 days at room temperature in moist chambers, approximately 42% of total DNA was degraded. Only 30% of these DNA samples still showed good RFLP patterns. However, no obvious relation between qualities of DNA analyzed by detection of RFLP and quantities of total and high-MW DNA became apparent. A case of familial relationship was ascertained by DNA fingerprints. Since DNA of good quality can be recovered from muscle tissues in large quantities, DNA extraction from muscle tissues and detection of RFLP patterns should be very useful for individual identification in autopsy cases.

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.

The use of hypervariable DNA minisatellite probes recognizing repetitive genomic DNA sequences has become a valuable and powerful tool in paternity testing as well as in forensic stain analysis (Jeffreys et al. 1985, 1987; Werrett et al. 1988). It has been shown that bacteriophage Ml3 DNA can also be used to obtain hypervariable DNA restriction fragment patterns in humans and other species (Vassart et al. 1987). To obtain more informative and specific fragment patterns for the DNA ‘fingerprint’ analysis in man, we have used Ml3 DNA as a probe to screen a human genomic library. Thus, we have isolated the minisatellite DNA probe MZ 1.3 (Schacker et al., in press). MZ 1.3 is a 1.9 kb fragment containing a 27 bp core sequence which is repeated 40 times. Alignment of the MZ 1.3 repeat with the 15 bp repeat of the M13 protein III gene has revealed identity at 8–11 positions, i.e. 53 – 73%. No significant homology >70% was found by comparison of MZ 1.3 to the core repeat sequences of 33.6 and 33.15 described by Jeffreys et al. (1985).

A common and revised nomenclature of the allotypes of the fourth component (C4) of human complement has been proposed. It is based on the results of the C4 Reference Typing of the VIth Complement Genetics Workshop and Conference, Mainz, FRG, 1989, the previous C4 nomenclature and the guidelines for human gene nomenclature (ISGN). The designation of allotypes derives from their relative electrophoretic mobility, the distinction between C4A and C4B proteins from their relative hemolytic activity. Common alleles retain their single digit numeric designation, intermediate variants their two- or three-digit designations; newly discovered alleles should not interfere with already described variants. At least 13 C4A alleles, 16 C4B alleles as well as non-expressed genes at each C4 locus are presently known. There are also duplicated loci of each C4 gene; they should be designated by repetition of the locus symbol at the haplotype or genotype level. As a phenotype they will be placed in parenthesis without repetition of the locus symbol. Aberrant allotypes or hybrid genes should be explained by a special suffix. No special nomenclature is recommended for restriction fragment length polymorphisms. Their designation should follow the general rules of the ISGN.

Genetic linkage between the HL-A and Bf loci could be confirmed in 43 families with 168 offspring. In 4 families, 5 recombinants out of 82 informative meiotic divisions were observed (r = 6.1%). The localisation of the Bf marker system was studied in 3 families with crossovers between HL-A and MLC. From these data the following map order of human chromosome 6 can be proposed: HL-A (1st locus) -- HL-A (2nd locus)--MLC-Bf---PGM(3). The fact that important components of the classical and alternate pathway of complement activation are governed by genes closely linked with HL-A and MLC loci leads to the proposition to include the Bf system into the Major Histocompatibility Complex in man.

Wir haben Thrombozytenkonserven von 109 und Thrombozyten-“pellets” aus Plasma von 126 Spendern aus dem Koln-Bonner Raum mit der Agarosegel-Elektrophorese mit nachfolgender Immunfixation auf den Faktor XIIIA-Polymorphismus untersucht. Die gefundenen Allelfrequenzen fur FXIIIA*1 von 0.7787 und FXIIIA*2 von 0.2212 stehen in guter Ubereinstimmung mit anderen, fur die weise Rasse erhobenen Frequenzen. Der FXIIIA-Polymorphismus kann ohne Bedenken in die Abstammungsbegutachtung einbezogen werden.

The Dw1 specificity was highly correlated with the serologically determined HLA-DR1 antigen in the Eighth International Histocompatibility Workshop 1980. By testing a large number of HLA-Dw1, DR1 defined homozygous typing cells (HTC) in a checkerboard primary mixed lymphocyte reaction, on a panel of about 30 HLA-DR1 heterozygous individuals, and in family segregation, three Dw1 "subtypes" could be defined in association with certain HLA-A, -B, and -C-antigens. HTC TA, FRI, and FRA carrying the HLA haplotypes A11, B35, Cw4 or A3, B35, Cw4 in the homozygous state gave positive typing results with most HLA-DR1 positive panel members and stimulated only four other Dw1 HTCs (SRR less than or equal to 35%). In contrast to this operationally "broad" specificity, Dw1-HTC-HEN (HLA-A2, B44, C-, homozygous) was non-stimulatory to all HTCs except one, but gave high responses against these, leading to the definition of a "narrow" specificity included in the "broad" one. Another such "narrow" specificity was represented by HTC FEE (HLA-A2, B27, Cw2 homozygous). Typing patterns with FEE were mostly different from those defined with other HTC. In family studies a specific typing pattern for this HTC could be shown to segregate with HLA. However, within some of these responses a contribution of the HLA haplotype in the trans position must be assumed.

Summary With an improved method for extraction of HL-A soluble antigen from dried blood stains, eighteen HL-A antigens were tested in the microcytotoxicity test as described in the preceding paper (Rittner & Waiyawuth, 1974). With the antisera available, seven antigens could not be detected with sufficient confidence. As a serious handicap of the microcytotoxicity test, a selected panel of donor lymphocytes must be permanently available. A comparison between the microcytotoxicity and the microcomplement fixation test performed on platelet material recovered from the stains led to the conclusion that the microcomplement fixation test is better suited for a routine laboratory. Encouraging results were obtained in a larger series of microcomplement fixation tests with white blood cells from dried blood stains. Significant chi-squared values were calculated for fourteen of sixteen antigens tested. The possible causes of false positive and negative reactions are discussed. Anticomplementary reaction of anti-HL-A sera in microcomplement fixation tests should be taken into account.

Since the estimation of recombination fractions is only an arbitrary means to map genetic loci on chromosomal regions, family studies of cases with informative cross-overs within the region in question are of decisive importance. This paper reports the study of a family with a HLA-B to HLA-D cross-over which is informative in the Bf system. The possible reasons for conflicting published data on Bf gene mapping are discussed.

Population and family data collected at the Reference Laboratory for the polymorphism of the C3 complement component of man support the view that there is very little, if any, gene frequency variation among Caucasian populations. The observed segregation ratios are in close agreement with the expected ratios. Three ‘new’ variants are reported in 1973/74: ‘F 0.9’, ‘S 1.0’ and ‘S 1.6’. Since rare variants cannot be designated in terms of absolute mobilities, the recommendation of the First International Symposium and Workshop at Bonn in 1972 is renewed to compare ‘new’ rare variants with reference standard sera in the Reference Laboratory. The common C3 polymorphism is also detected in the C3c conversion product as revealed by the Pt typing technique. Rare variants, however, are not reliably determined in this method.

HL-A antigens were determined in 41 unrelated coeliac children and in clinically healthy parents and siblings of 40 of these patients using a lymphocyte microcytotoxicity test. 58.5% of the coeliac patients had phenotype HL-A 8 compared with an HL-A 8 frequency of 16.6% in a control group of 320 unrelated individuals (P less than 0.0005). Excluding five patients not of pure German origin HL-A 8 frequency increases to 63.9%. The increase of HL-A 1 frequency in coeliac patients is attributed to linkage disequilibrium between HL-A 8 and HL-A 1. The haplotype HL-A 1.8 frequency was significantly increased in coeliac children (P less than 0.0001) with frequency elevation also in parents (P approximately 0.025) but not in siblings. Furthermore, an increase in frequency of HL-A 12 and a decreased frequency of HL-A 7 and HL-A 9 was found in coeliac patients. Five clinically healthy siblings had the same HL-A haplotypes as their affected sisters and brothers.