[Responsible dealing with sexuality. Recommendations in a clinical institution]. / Verantwortlicher Umgang mit Sexualität. Empfehlungen in einer klinischen Einrichtung.

Sexuality is excluded in house regulations, guidelines, instructions and regulations in German hospitals. The English literature does not show much more, but more often the wish for clear guidelines is formulated. Under the guidance of the clinical Ethics Committee a paper with recommendations was prepared, which comprises regulations for responsible handling of sexuality in the Pfalzklinikum. This includes sexuality of acute patients in psychiatry, nursing home inhabitants, forensic patients and above all patients in the department of child and youth psychiatry. The right of self-realization on the one hand, the staff's responsibility for patients with limitations in the determination of one's intent on the other hand and the rules for staff members define the range of the paper.

Null alleles of human complement C4. Evidence for pseudogenes at the C4A locus and for gene conversion at the C4B locus.

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.

Genetic basis of human complement C4A deficiency. Detection of a point mutation leading to nonexpression.

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.

Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants.

Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease.

A TNF-alpha promoter polymorphism is associated with juvenile onset psoriasis and psoriatic arthritis.

Tumor necrosis factor-alpha is considered to be one of the important mediators in the pathogenesis of psoriasis. A strong association of juvenile onset psoriasis with the major histocompatibility complex encoded HLA-Cw6 antigen has been reported but it is unclear whether Cw6 itself or a closely linked gene is involved in the pathogenesis. This study has focused on the association of promoter polymorphisms of the major histocompatibility complex encoded tumor necrosis factor-alpha gene with psoriasis and psoriatic arthritis. Tumor necrosis factor-alpha promoter polymorphisms were sought by sequence-specific oligonucleotide hybridization and by direct sequencing in Caucasian patients with juvenile onset psoriasis and with psoriatic arthritis and in healthy controls. A mutation at position -238 of the tumor necrosis factor-alpha promoter was present in 23 of 60 patients (38%; p < 0.0001; p[corr] < 0.008) with juvenile onset psoriasis and in 20 of 62 patients (32%; p < 0.0003; p[corr] < 0.03) with psoriatic arthritis, compared with seven of 99 (7%) Caucasian controls. There was a marked increase of homozygotes for this mutation in the psoriasis group. Another mutation at position -308 was found in similar proportions of patients and controls. Our study shows a strong association of the tumor necrosis factor-alpha promoter polymorphism at position -238 with psoriasis and psoriatic arthritis. Our findings suggest that this promoter polymorphism itself or a gene in linkage disequilibrium with tumor necrosis factor-alpha predispose to the development of psoriasis.

Sandwich enzyme-linked immunosorbent assays for the quantification of the C4 isotypes (C4A and C4B) in human plasma.

Sandwich enzyme-linked immunosorbent assays were developed to determine the concentration of the isotypes of the fourth component of human complement (C4A and C4B) in human plasma. In the case of C4A a monoclonal antibody against a common determinant of the alpha chain was used to capture the protein. The bound antigen was then detected with a biotinylated monoclonal antibody reacting exclusively with the C4A isotype, followed by peroxidase labeled avidin. For the quantification of C4B, C4B-specific monoclonal antibodies were coated onto a microtitration plate in order to capture the protein. Bound antigen was then detected with a biotinylated monoclonal antibody directed against C4 followed by peroxidase labeled avidin. The assays, which were rapid, selective and specific for C4A and C4B, respectively, provide an alternative to gel electrophoresis and blot procedures for the study of unexpressed alleles ('null alleles') at each of the C4 loci.

Polymerase chain reaction analysis of the Xba I polymorphism of the human complement C4 genes provides evidence for strong haplotype conservation.

The genes coding for the two isotypes of the fourth component of human complement, C4A and C4B, are located between the HLA-B and -DR loci of the MHC. We studied the linkage relationship of the previously described XbaI RFLP to obtain further insight into the evolution of the tandemly arranged C4 genes. Using exon-specific PCR amplification followed by restriction analysis and direct DNA sequencing, the polymorphic site could be located in exon 40 of the C4 gene (cDNA position 5095). The polymorphism does not change an amino acid residue. Using nested PCR amplification with isotype-specific primers to amplify either C4A or C4B alleles the haplotype arrangement of the XbaI sites in both isotypic C4 genes was analyzed independently. It was observed that the XbaI restriction site was either present or absent in both C4 genes of a given haplotype. In a study of 106 Caucasian haplotypes, only two different haplotypes could be identified carrying a C4A gene with and a C4B gene without the XbaI restriction site. Also, the XbaI site could only be detected in long C4 genes possessing the 6.5-kb insertion in intron 9. Our findings provide evidence that the mutation creating the XbaI polymorphism occurred in an ancestral C4 gene already carrying the long intron 9. The duplicating resulting in the presence of two isotypic genes, C4A and C4B, must have taken place subsequently giving rise to haplotypes with or without the XbaI site.

TAP-polymorphisms in juvenile onset psoriasis and psoriatic arthritis.

Juvenile onset psoriasis is strongly associated with the HLA-class I genes Cw6 and B57 whereas patients with psoriatic arthritis show an increased frequency of HLA-B27. It is unclear whether additional major histocompatibility genes also increase disease susceptibility. The TAP genes (transporter associated with antigen processing) encode two membrane-spanning proteins that translocate antigenic peptides from the cytoplasm into the endoplasmic reticulum. Comparison of 60 patients with juvenile onset psoriasis, 63 psoriatic arthritis patients, and 101 caucasoid controls revealed an increase of the TAP1*0101 allele in the psoriasis group, that could not be explained by linkage to other investigated HLA genes. There were no differences for TAP2 alleles.

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.

Quantitative competitive PCR as a technique for exploring flea-Yersina pestis dynamics.

We used a quantitative competitive polymerase chain reaction assay to quantify Yersinia pestis loads in fleas and bacteremia levels in mice that were used as sources of infectious blood meals for feeding the fleas. Xenopsylla cheopis, the Oriental rat flea, achieved higher infection rates, developed greater bacterial loads, and became infectious more rapidly than Oropsylla montana, a ground squirrel flea. Both flea species required about 10(6) Y. pestis cells per flea to be able to transmit to mice. Most fleas that achieved these levels, however, were incapable of transmitting. Our results suggest that at the time of flea feeding, host blood must contain > or = 10(6) bacteria/ml to result in detectable Y. pestis infections in these fleas, and > or = 10(7) bacteria/mL to cause infection levels sufficient for both species to eventually become capable of transmitting Y. pestis to uninfected mice. Yersinia pestis colonies primarily developed in the midguts of O. montana, whereas infections in X. cheopis often developed simultaneously in the proventriculus and the midgut. These findings were visually confirmed by infecting fleas with a strain of Y. pestis that had been transformed with the green fluorescent protein gene.

LMP polymorphisms do not influence disease expression in psoriatic arthritis.

OBJECTIVES: To investigate the potential role of the HLA-linked LMP2 (low molecular weight protein) and LMP7 gene polymorphisms in conjunction with HLA class I and class II genes on the disease pattern in individuals with psoriatic arthritis (PsA). METHODS: Sixty-three patients with PsA and 99 unrelated controls were typed for HLA-class I and II antigens. LMP2 and LMP7 polymorphisms were determined by PCR and subsequent single stranded conformation polymorphism (SSCP) analysis or restriction enzyme digestion. RESULTS: PsA was associated with B27 (p < 0.0004), B57 (p < 0.002) and Cw2 (p < 0.008). Spondylarthritis was strongly associated with HLA-B27 (p < 0.000001), Cw2 (p < 0.0003) and DR4 (p < 0.008). For the polyarthritic pattern the only association was with B57 (p < 0.007). There was no association between the LMP2 or LMP7 genotypes and any particular disease pattern. CONCLUSIONS: These findings do not support an involvement of the HLA linked LMP2 and LMP7 gene polymorphisms in disease expression in psoriatic arthritis in addition to the known HLA class I and II associations.

[The identification of unknown corpses by x-ray comparison]. / Identifizierung unbekannter Leichen durch Röntgenbildvergleich.

Between 1987 and 1993, 30 unknown bodies were identified by means of pre- and postmortem radiographs at the Forensic Institute in Mainz. Our experience indicates that radiological identification comprises a useful, rapid and cheap method, at least as valuable as dactyloscopy or odontological comparisons. The ages of available radiographs were up to 25 years; the most suitable regions are the skull (11), pelvis and lumbar spine (6), lower femur and knee (5) and distal leg with the ankle joint (5). In respect of the problem of objectifying the probability of identity, possibilities for solutions are shown.

Medical law and ethics in abortion--results of a comparative study in Japan and Germany.

Based on interviews with 180 women in Ube, Japan, and 200 woman in Mainz, Germany, the authors report on different opinions on reproductive medicine, with particular reference to abortion. In the light of the historical German-Japanese legal relationship, the latest ruling of the Bundesverfassungsgericht on abortion is discussed, with some remarks from J. Reiter on its social ethics.

Application of mtDNA sequence analysis in forensic casework for the identification of human remains.

In four forensic cases of unidentified skeletal remains investigated in the last year, we were able to attach three to missing persons. In one case we could show that the discovered bone sample did not fit to a missing child. The method for mitochondrial DNA analysis for the routine identification of skeletal remains was established in our institute by typing bone samples of defined age obtained from Frankfurt's cemetery. Reproducible results were obtained for bones up to 75 years old. For analysis the bone samples were pulverised to fine powder, decalcified and DNA was extracted. From the DNA we amplified a 404-bp fragment from HV-1 and a 379-bp fragment from HV-2 of the mtDNA control region. After sequencing of the PCR products, the results were compared to the Anderson reference sequence and to putative maternal relatives.

Use of the software 'Poser4' in reconstruction of accident and crime scenes.

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.

Computer-assisted simulation of conventional roentgenograms from three-dimensional computed tomography (CT) data--an aid in the identification of unknown corpses (FoXSIS).

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, centering of the X-ray beam, the location of the object in the patch 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.

Genetic analysis of the short tandem repeat system D12S391 in the German and three Asian populations.

Genomic DNA samples from 222 individuals from Southern China, 154 individuals from Thailand, 100 individuals from Japan as well as from 124 German individuals were analysed for the short tandem repeat (STR) locus D12S391. Typing was carried out by polymerase chain reaction (PCR) amplification and subsequent polyacryramide gel electrophoresis and silver staining. In total, 12 alleles could be distinguished in two of the populations. Among Chinese, allele 19 is the most common with a frequency of 0.225, and among Germans, allele 18 with a frequency of 0.186. In the Thai population only 11 alleles could be distinguished and allele 19 is the most common with a frequency of 0.198. In Japanese, two previously unknown alleles 27 and 28 were detected, 14 alleles could be distinguished, and allele 18 is the most common with a frequency of 0.295. The expected exclusion chance for Chinese, Thai, Japanese and Germans in paternity cases is 0.67, 0.71, 0.67 and 0.75, respectively, and the discrimination power in identification cases is 0.95, 0.96, 0.95 and 0.97, respectively. Testing of the observed genotype distributions for Hardy-Weinberg equilibrium did not reveal any significant deviations. Segregation studies of 124 meioses among German families did not reveal any mutations at the D12S391 locus. In casework studies two variant alleles were detected with a trimeric repeat each (17.3 and 18.3), which have been confirmed by sequencing.

Tandem repeat structure of the duplicated Y-chromosomal STR locus DYS385 and frequency studies in the German and three Asian populations.

The Y-chromosomal short tandem repeat (STR) locus DYS385 can be typed using PCR amplification and separation of the resulting polymorphic fragments by non-denaturing high resolution polyacrylamide gel electrophoresis followed by silver staining. The PCR primers amplify a duplicated repeat sequence on the Y chromosome revealing a two-band pattern in male individuals. To determine the internal repeat structure as a basis for a consensus nomenclature, DNA sequence analysis was carried out after subcloning of PCR-amplified fragments revealing the uniform 4-bp repeat structure 'GAAA'. The shortest allele observed consisted of 10 repeat units thus providing the basis for the designation 'allele 10'. Except for isolated point mutations, no systematic differences could be observed either in the repeat sequence or in the flanking regions between the two fragments of a given individual. Thus it was not possible to discriminate between the two loci of the DYS385 system. Four population samples of German (n = 146), Chinese (n = 100), Japanese (n = 100), and Thai (n = 95) origin were studied. In the four groups, alleles 10 to 24 could be observed and genotype frequencies differed significantly. In Germans only one common genotype was present (11-14; 33.8% frequency). In the Asian populations, the frequencies were more evenly distributed with the 13-13 genotype (9%) in Chinese, the 13-17 genotype (14%) in Japanese and the 14-18 genotype (7%) in Thai being the most common. Overall, 69 different genotypes were found, of these 36 were observed in Germans, 36 in Chinese, 33 in Japanese and 44 in Thai. No mutations were detected in 62 father-son pairs. Thus DYS385 is a highly polymorphic STR system with population-specific genotype distributions.

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' 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/Pvu II, 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.

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.