Complement component C3: molecular basis of the C3*S025 variant and evidence for molecular heterogeneity of other variants.

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 mutat…

Application of DNA techniques for identification using human dental pulp as a source of DNA

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 D…

Hepatitis B surface antigen presentation and HLA-DRB1*– lessons from twins and peptide binding studies

Summary The aim of this study was to investigate the underlying mechanisms of the genetic association between certain HLA-DRB1* alleles and the immune response to HBsAg vaccination. Therefore, HBsAg peptide binding to HLA-DR molecules was measured in vitro by peptide binding ELISAs. Additionally, HBsAg-specific T cell reaction and cytokine profile of immune response were analysed ex vivo in ELISPOT assays and DR-restriction of T-cell proliferative responses was investigated with HBsAg specific T cell clones. In addition, we compared HBsAg specific T cell responses of 24 monozygotic and 3 dizygotic twin pairs after HBsAg vaccination. Our results showed that the peptide binding assays did not…

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

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 allotyp…

Tumor necrosis factor alpha promoter polymorphism at position -238 is associated with chronic active hepatitis C infection

Tumor necrosis factor alpha (TNF-alpha) is involved in the pathogenesis of chronic hepatitis C virus infection. The gene for TNF-alpha is encoded in the major histocompatibility locus (MHC). Two polymorphisms at positions -308 and -238 in the TNF-alpha promoter region might influence TNF-alpha expression. These promoter polymorphisms have been linked previously to a number of infectious diseases. TNF-alpha promoter polymorphisms at positions -238 and -308 were studied by DNA sequencing and sequence-specific oligonucleotide hybridization in 82 individuals with chronic hepatitis C and 99 control subjects. Subjects had been HLA class I and class II typed in a previous study. The frequency of t…

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 1…

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. …

TAP-polymorphisms in juvenile onset psoriasis and psoriatic arthritis.

Abstract 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 o…

Introduction of an single nucleodite polymorphism-based “Major Y-chromosome haplogroup typing kit” suitable for predicting the geographical origin of male lineages

The European Consortium "High-throughput analysis of single nucleotide polymorphisms for the forensic identification of persons--SNPforID", has performed a selection of candidate Y-chromosome single nucleotide polymorphisms (SNPs) for making inferences on the geographic origin of an unknown sample. From more than 200 SNPs compiled in the phylogenetic tree published by the Y-Chromosome Consortium, and looking at the population studies previously published, a package of 29 SNPs has been selected for the identification of major population haplogroups. A "Major Y-chromosome haplogroup typing kit" has been developed, which allows the multiplex amplification of all 29 SNPs in a single reaction. A…

Preparation of degraded human DNA under controlled conditions

Abstract DNA typing through analysis of short tandem repeats (STRs) and mitochondrial DNA (mtDNA) by means of the polymerase chain reaction (PCR) and sequencing are the common methods for the forensic identification of persons and reconstruction of kinship, especially when skeletal human remains have to be analyzed. Furthermore, samples typically found at crime scenes may be both quantitatively and qualitatively inadequate since they may contain very scarce and often degraded DNA due to exposure to heat, light, humidity, and microorganisms. In order to improve the performance of STR typing technology in those cases where DNA availability is limited, it would be desirable to have a source of…

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, nonresponsiven…

Forensic mtDNA hair analysis excludes a dog from having caused a traffic accident

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 fragm…

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

Abstract 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 Ja…

The eighth component of human complement: molecular basis of C8A (C81) polymorphism.

Using an exon-specific polymerase chain reaction (PCR) followed by direct DNA sequence analysis we have analyzed the polymorphism of the alpha-chain of the eighth component of human complement (C8) at the DNA level. We found that two common alleles, C8A*A and C8A*B, are characterized by the substitution of a single amino acid (Gln to Lys), which is caused by a point mutation of a single nucleotide (C to A) in exon 3 at position 187 of the mature C8 alpha cDNA sequence. Based on this mutation, an allele-specific PCR was designed detecting the two alleles of C8A. We applied this method to type the C8A polymorphism using DNA samples from a Chinese Han population. The comparison with the data o…

Expert witness in paternity testing in Germany.

In Germany, paternity testing can be ordered by a judge as well as by private persons. In the case of private counselling, the expert has the duty to inform the parties on all medical, legal and ethical aspects. Informed consent must be given by each individual included in an exploration of family relationships. Due to the rapid progress of DNA typing in even minute amounts after polymerase chain reaction amplification, genotypes of short tandem repeat systems can be elicited by extraction from single cells. Therefore, the number of unlawful investigations of paternal relationships is steadily increasing. Here the requirements for paternity testing laboratories and sanctions for unlawful ex…

Application of DNA Polymorphisms in Paternity Testing in Germany: Solution of an Incest Case Using Bacteriophage M13 Hybridization with Hypervariable Minisatellite DNA

More than 25 blood, serum, and enzyme polymorphisms have been introduced into paternity testing in Germany in recent years (Rittner, 1975). If a “no” decision is defined by exclusion, and a “yes” decision requires a probability of 99.73 % or more, more than 90 % of court cases can be solved in this respect. A few cases not being clarified by a standard expertise include: 1) Cases with more than one alleged man if the men and/or the mother and the men are related. 2) Some cases where the putative father is deceased, and neither the parents nor the legitimate offspring are available for the study. 3) Cases where possible exclusion in a given polymorphic system interferes with an overall evide…

Reliability of Restriction Enzyme Digestions of Genomic DNA for the Generation of DNA Fingerprints

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…

Cholinesterase variants: rapid characterisation by PCR/SSCP and evidence for molecular homogeneity.

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 populati…

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. …

Changes in the DNA profile caused by toxic substances?

Minisatellite DNA Probe MZ 1.3: Application in Paternity Testing and Estimate of the Number of Genetic Loci

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 …

Considerations from the European DNA profiling group (EDNAP) concerning STR nomenclature

(1) The nomenclature of any STR follows from comparison with a control allelic ladder; availability of reference allelic ladders is central to any scheme. The components of an allelic ladder should be sequenced. (2) The DNA commission recommended a nomenclature based upon the number of repeat sequences present in an allele. Whereas this method is suitable for typing simple STRs, complex hypervariable repeats such as ACTBP2 do not conform to a simple repeating structure. We propose that designation of complex STR repeats such as ACTBP2, D11S554 and APOAI1 follows from the size of specific alleles. Because the size is dependant upon the primers utilised, the size is not definitive (it may als…

Characterization of non-expressed C4 genes in a case of complete C4 deficiency: identification of a novel point mutation leading to a premature stop codon.

The genetic basis of complete C4 deficiency in a patient with SLE was investigated. Previous studies have demonstrated that this patient has two different major histocompatibility complex (MHC) haplotypes that each contain a major deletion and a non-expressed C4 gene. In the present study, non-expression of the C4 genes was explained by the finding of two distinct C4 gene mutations. A previously described two base pair insertion in exon 29 of the C4 gene was detected in the paternal MHC haplotype [HLA-A2, B40, SC00, DR6]. The maternal haplotype [HLA-A30, B18, F1C00, DR3] carried a C4 gene with a one base pair deletion in exon 20 generating a premature stop codon. This mutation was neither f…

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

Abstract 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 con…

Quantitative and qualitative analysis of DNA extracted from postmortem muscle tissues

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 goo…

Rapid microarray-based typing of forensic SNPs

The single base extension-tag array (SBE-Tag Array) method is carried out on glass slides and combines the specificity of minisequencing for SNP typing with the high throughput capacity of microarrays. Following multiplex PCR, a single tube SBE reaction is carried out, and the fluorescent labelled extension products are hybridized to the complementary DNA sequence tag (cTag) immobilized on a glass slide for locus-specific laser scan analysis. The aim is to prove and optimise the conventional microarray reaction on accuracy and efficiency for forensic applications. © 2005 Elsevier B.V. All rights reserved.

Report of the European DNA profiling group (EDNAP)-an investigation of the hypervariable STR loci ACTBP2, APOAI1 and D11S554 and the compound loci D12S391 and D1S1656

This paper describes the results of three collaborative exercises which continues the EDNAP theme to explore whether uniformity of DNA profiling results could be achieved between European laboratories using STRs. In an earlier exercise, complex hypervariable AAAG-repeat STR loci were investigated, but reproducibility was found to be poor because of the variation of techniques used by participating laboratories. In the exercise reported here, an internal allelic ladder composed of ACTBP2 and D11S554 fragments was distributed. This ladder was used to size ACTBP2 analysed by a "singleplex" PCR amplification and D11S554 combined with APOAI1 in a separate "duplex" reaction. Laboratories were ask…

Forensic validation of the SNPforID 52-plex assay.

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations. The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) react…

Results of collaborative study regarding the standardization of the Y-linked STR system DYS385 by the European DNA Profiling (EDNAP) group.

Y-chromosome linked short tandem repeat (STR) loci are inherited as a closely linked haplotype, which appears to remain stable in a given paternal lineage over many generations. In forensic cases, Y-linked STRs are particularly useful for the identification of human remains as well as in rape cases with mixed male/female stain samples. DYS385 is derived from tandemly duplicated segments of the Y chromosome thus giving rise to two fragments of variable length which do not behave like alleles but genotypes. The European DNA Profiling (EDNAP) group has carried out a collaborative exercise among 14 participating laboratories using DYS385 for typing of five unknown bloodstains and a control samp…

Application of Nanogen microarray technology for forensic SNP analysis

Abstract The NanoChip® Molecular Biology Workstation using electronic microarrays is an approach for rapid and high throughput analysis of SNPs. This instrument is fully automated and uses a microchip for electronic addressing of capture probes to specific array sites followed by electronic hybridisation of the single stranded PCR products, and passive hybridisation of fluorescently labelled reporter probes. Discrimination is achieved by applying thermal stringency to denature the mismatched reporters. 48 SNP assays have been designed using the ‘capture down’ assay which applies a thermal ‘touch down’ strategy to obtain the best reporter probe discrimination.

Online reference database of European Y-chromosomal short tandem repeat (STR) haplotypes

The reference database of highly informative Y-chromosomal short tandem repeat (STR) haplotypes (YHRD), available online at http://ystr.charite.de, represents the largest collection of male-specific genetic profiles currently available for European populations. By September 2000, YHRD contained 4688 9-locus (so-called "minimal") haplotypes, 40% of which have been extended further to include two additional loci. Establishment of YHRD has been facilitated by the joint efforts of 31 forensic and anthropological institutions. All contributing laboratories have agreed to standardize their Y-STR haplotyping protocols and to participate in a quality assurance exercise prior to the inclusion of any…

Development of a pentaplex X-chromosomal short tandem repeat typing system and population genetic studies

Quadruplex and pentaplex systems for polymerase chain reaction amplification of X-chromosomal short tandem repeats DXS101, HPRTB, DXS8377, DXS981 (STRX1) and DXS6789 were developed for automated profiling of liquid and membrane-bound DNA samples. Chinese, Japanese and Thai populations were typed using a quadruplex system, while German and Philippine populations were analyzed using a five-locus system. Out of 88 meioses studied in Philippine family samples at each locus, a possible one repeat deletion (allele 51 to 50) at DXS8377 was observed in a father-daughter pair. Exact tests performed on genotype data from females in the Philippine, German and Thai populations indicated that these grou…

Achievement of Interlaboratory Uniformity — A Summary of Work Carried out by the EDNAP Group

This paper describes a collaborative exercise intended to demonstrate whether uniformity of DNA profiles results could be achieved between different European laboratories. It was shown that this goal would be obtained provided that a common protocol was followed (specifically the use of a common electrophoretic buffer is the most important parameter).

The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition

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 tr…

C4A deficiency and nonresponse to hepatitis B vaccination

Hepatitis B vaccination failure has been linked to the presence of certain human leukocyte antigen class II alleles. However, the functional background of these associations has remained unclear. Complement component C 4 is encoded within the major histocompatibility complex and is essential for classical pathway activation.Healthy individuals (n=4269) were vaccinated in a prospective trial with Engerix B. Nonresponse was classified as anti-HBs10 U/l after the last vaccination. Seventy-three nonresponders (NR) (1.7%) were identified. For comparison 53 responders (R) (anti-HBs10 IU/l) were drawn randomly from the same cohort. C4 allotyping was carried out by high-voltage agarose gel electrop…

Heterogeneity in the genetic basis of human complement C9 deficiency

Major histocompatibility complex (MHC) class III genetics in two Amerindian tribes from southern Brazil: the Kaingang and the Guarani.

Population genetic studies of the major histocompatibility complex (MHC) class III region, comprising C2, BF and C4 phenotypes, and molecular genetic data are rarely available for populations other than Caucasoids. We have investigated three Amerindian populations from Southern Brazil: 131 Kaingang from Ivaí (KIV), 111 Kaingang (KRC) and 100 Guarani (GRC) from Rio das Cobras. Extended MHC haplotypes were derived after standard C2, BF, C4 phenotyping and restriction fragment length polymorphism (RFLP) analysis with TaqI, together with HLA data published previously by segregation analysis. C2 and BF frequencies corresponded to other Amerindian populations. C4B*Q0 frequency was high in the GRC…

The human complement C9 gene: structural analysis of the 5′ gene region and genetic polymorphism studies

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 we…

A multiplex assay with 52 single nucleotide polymorphisms for human identification.

A total of 52 SNPs reported to be polymorphic in European, Asian and African populations were selected. Of these, 42 were from the distal regions of each autosome (except chromosome 19). Nearly all selected SNPs were located at least 100 kb distant from known genes and commonly used STRs. We established a highly sensitive and reproducible SNP-typing method with amplification of all 52 DNA fragments in one PCR reaction followed by detection of the SNPs with two single base extension reactions analysed using CE. The amplicons ranged from 59 to 115 bp in length. Complete SNP profiles were obtained from 500 pg DNA. The 52 loci were efficiently amplified from degraded samples where previously on…

HLA-DRB1*1301 AND *1302 protect against chronic hepatitis B

Abstract Background/Aims: The outcome of acute hepatitis B infection may be influenced by host factors like the major histocompatibility complex (MHC). We have investigated MHC class I and class II antigens in patients with chronic hepatitis B compared to a healthy control population. To confirm the findings of this first study we performed a second study in a group of subjects who had spontaneously recovered from acute hepatitis B infection. Methods: Frequencies of MHC class I and class II antigens were analyzed in patients with chronic hepatitis B virus infection and in control subjects. MHC class I typing was done by standard microlymphocytotoxicity assays. DRB1 and DQA1 genotypes were d…

PCR Typing of Alu Elements — Molecular Genetics and Forensic Application

Alu repeats belong to the family of short interspersed elements (SINEs) and are among the most abundant repetitive DNA sequences in the mammalian genome. They represent mobile genetic elements ancestrally derived from the 7SL RNA gene and have presumably spread within the genome by retroposition (reviewed in [1]). A particular group of Alu repeats appears to be human-specific (HS subfamily) and has expanded only recently within the human genome as indicated by distinct dimorphisms at various loci due to the presence or absence of an Alu repeat. In recent extensive studies, the frequency distributions of Alu insertions at selected loci in various human racial groups and populations were dete…

A NEW PCR-BASED TYPING OF THE RODGERS AND CHIDO ANTIGENIC DETERMINANTS OF THE FOURTH COMPONENT OF HUMAN COMPLEMEMT

The Rodgers (Rg) and Chido (Ch) blood groups are antigenic determinants of the fourth component of human complement C4. They are associated with the two isotypes of C4, C4A and C4B, respectively. They serve as markers to distinguish C4A from C4B as well as for the definition of subtypes of common and rare allotypes. As an alternative to the serological typing method using human alloantisera, a PCR typing procedure with sequence-specific primers (PCR-SSP) was designed. The method was tested on selected DNA samples from individuals with well-defined C4 allotypes. No false-positive or false-negative typing results were obtained and all the determinant combinations could be distinguished. The P…

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…

HLA DRw8 and primary biliary cirrhosis

A multiplex SNP typing approach for the DNA pyrosequencing technology

Abstract We have developed a multiplex Pyrosequencing assay which enables the simultaneous analyses of 23 single nucleotide polymorphisms (SNPs) from the human genome selected by the SNPforID Consortium. In our investigations we have studied the multiplex capacity of the PSQ™ 96MA instrument (Biotage AB). To test the reliability of SNP typing by Pyrosequencing the SNPs were analysed in parallel by using the SNaPshot minisequencing technique as reference method.

Polymorphism of the Complement C8A and -B Genes in Two Families with C8β Deficiency and Neisserial Infections

Serum samples from members of two Italian families with complement C8 beta deficiency were studied by SDS-PAGE under nonreducing conditions and by IEF. The proband of family I had suffered from two episodes of purulent meningitis and two of her uncles had suffered from only one episode, while the proband of family II had suffered from three different episodes. In contrast to previous findings, where C8 beta deficiency was cosegregating with C8A (alpha-gamma) allotype A, the proband of family II had the C8A allotype B. In addition, in one of her sons a novel variant of the C8 beta chain was detected. Studies at the DNA level in family I, using a recently described PCR system, demonstrate the…

Fingerprints from fingerprints

Besides ‘‘classical’’ biological materials such as blood and sperm, epithelial cells from latent fingerprints are targeted in forensic sciences. In addition to studies using latent fingerprints applied to beer glasses [1], T-shirts left on crime scenes [2] and various other objects [3], we report the detection of STR profiles from latent fingerprints deposited on ordinary sheets of paper. In contrast to the relatively high number of epithelial cells from saliva or from excessively pressured fingerprints during strangulation [4,5], the experiments with latent fingerprints are expected to generate only a very small number of epithelial cells. Moreover, cells remaining on objects touched only …

STR analysis of artificially degraded DNA--results of a collaborative European exercise.

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak heig…

Report of the European DNA profiling group (EDNAP): an investigation of the complex STR loci D21S11 and HUMFIBRA (FGA).

This paper describes a collaborative exercise which was intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using two complex short tandem repeat (STR) loci. The loci D21S11 and HUMFIBRA (FGA) were chosen because they are commonly used by different European laboratories. D21S11 has approximately 14 common alleles (f > 0.001), whereas HUMFIBRA has 19 common alleles. Laboratories were asked to test seven blood stains, one of which was a known control, and to report the results to the coordinating laboratory. The exercise demonstrated that complex STRs were amenable to standardisation.

Application of whole genome amplification for forensic analysis

Abstract Fundamental to most forensic analyses is the availability of genomic DNA of adequate quality and quantity. To perform a multitude of genetic analyses and assays requires a sufficiently large amount of template. However, DNA yield from forensic samples is frequently limiting the extent of genetic typing. A possible solution to overcome this “bottleneck” of forensic and paleoarcheological DNA analyses could be the amplification of the entire genomic DNA prior to locus specific PCR analysis. Whole Genome Amplification appears to be a promising tool to obtain sufficient DNA amounts from forensic samples of limited quantity.

Pseudo-exclusion from paternity due to maternal uniparental disomy 16.

The investigation of a case of disputed paternity revealed indirect exclusion of the alleged father in the haptoglobin system and in the DNA single-locus system D16S309/Hinf I (MS205). The paternity index for the non-exclusion systems was > 10(6). Since both exclusion systems (HP and MS205) are located on chromosome 16, we investigated 10 microsatellite loci covering this chromosome with 10-20 cM resolution. Analysis of the child's chromosome showed only alleles of maternal origin and lack of inheritance of paternal alleles for five informative loci. The markers close to the centromere of chromosome 16 were heterozygous, whereas distal loci were either heterozygous or homozygous for materna…

Comparison of Different Methods for the Calculation of Indices of Paternity

The qualitative decision about paternity in trio cases on the basis of DNA multilocus profiles is no problem. If there are more than 1 or 2 exclusion patterns (band present in child, which is neither present in mother or alleged father), the putative father has to be excluded. The problems arise, if, in the case of a non exclusion, one wants to quantify the evidence for paternity. Different statistics have been proposed for this purpose. This paper discusses three of these statistics and demonstrates their application to real data.

Analysis of 29 Y-chromosome SNPs in a single multiplex useful to predict the geographic origin of male lineages

Abstract The European Consortium “High throughput analysis of single nucleotide polymorphisms for the forensic identification of persons—SNPforID” has performed a selection of candidate Y-chromosome SNPs (single nucleotide polymorphisms) for making inferences on the geographic origin of an unknown sample. A “Major Y chromosome haplogroup typing kit” has been developed, which allows the multiplex amplification of 29 SNPs in a single reaction followed by a single base extension (SBE) reaction (minisequencing) and separation of the resulting extension products by capillary electrophoresis.

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 …

Genotyping of 9 STR systems in combination with 11 diallelic polymorphisms on the Y-chromosome by fragment analysis and minisequencing

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 po…

Towards a Physical Map of the HLA Complex

The human major histocompatibility (HLA) complex is located on the short arm of chromosome 6 in the 6p21.31→6p21.33 region (1,2). There are three clusters of genes, the HLA class I, II, and III regions. Whereas the class III loci are only moderately polymorphic (see (3) for review), the class I and II genes which encode cell surface glycoproteins show an extreme degree of polymorphism. There are a minimum of 17 class I loci (4) and at least 15 genes for class II alpha and beta chains (5). In addition, we (6) and others (7) have recently demonstrated that also the loci for tumor necrosis factor (TNFA) and lymphotoxin (TNFB) are part of the HLA complex [see also Ragoussis et al., this volume]…

Paternity Analysis Using the Multilocus DNA Probe MZ 1.3

The multilocus minisatellite DNA probe MZ 1.3 detects hypervariable restriction fragment patterns in genomic DNA of man and animals. It can be used for segregation analysis in cases of disputed paternity (Schacker et al., 1991; Rittner et al., 1991a), for identification purposes in forensic medicine and stain analysis (Ogata et al., 1990; Rittner et al., 1991b), as well as in animal breeding for pedigree analysis and verification of inbred strains (Hins & Gruber, 1991). Hypervariable fragment patterns can be generated by using frequently cutting restriction enzymes, e.g. Hinf I, Hae Ill, Msp I, Mbo I, and Rsa I. A non-radioactive system using the digoxigenin antidigoxigenin system may be us…

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise.

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot …

A TNF-α Promoter Polymorphism Is Associated with Juvenile Onset Psoriasis and Psoriatic Arthritis

Tumor necrosis factor-α 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-α gene with psoriasis and psoriatic arthritis. Tumor necrosis factor-α promoter polymorphisms were sought by sequence-specific oligonucleotide hybridization and by direct sequencing in Caucasian patients with juvenile onset …

Speech of the Minister of Justice of Rheinland-Pfalz (Germany) Peter Caeser at the occasion of the opening of the 14th Congress of the International Society of Forensic Haemogenetics

Paternity Testing Commission of the International Society of Forensic Genetics: recommendations on genetic investigations in paternity cases

The International Society for Forensic Genetics (ISFG) has established a Paternity Testing Commission (PTC) with the purpose of formulating international recommendations concerning genetic investigations in paternity testing. The PTC recommends that paternity testing be performed in accordance with the ISO 17025 standards. The ISO 17025 standards are general standards for testing laboratories and the PTC offers explanations and recommendations concerning selected areas of special importance to paternity testing. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

The effect of whole genome amplification on samples originating from more than one donor

Abstract In this study, the GenomiPhi™ DNA Amplification Kit (Amersham Biosciences) was used to investigate the potential of whole genome amplification (WGA) when considering samples originating from more than one donor. DNA was extracted from blood samples, quantified and normalised before being mixed in ratios of 1:1, 1:3, 1:7 and 1:15. Proportions were checked using standard STR analysis before being subjected to WGA. Once amplified using GenomiPhi™, the ratios were again checked. Relative proportions were found to be maintained in the 1:1 and 1:3 ratios following WGA; the observed peak ratios were found to match the expected peak ratios regardless of the starting concentration of DNA. W…

Ankylosing spondylitis in monozygotic twins: studies on immunological parameters

OBJECTIVE—To examine immunological parameters that might explain disease discordance in monozygotic twin pairs with ankylosing spondylitis (AS). METHODS—11 monozygotic twin pairs (nine with AS, two with undifferentiated spondyloarthropathy) were investigated. The peripheral T cell receptor Vβ repertoire was investigated using FACS analysis and 14 different Vβ antibodies. In addition serum samples were tested for antibodies to Klebsiella pneumoniae, Streptococcus pyogenes, Candida albicans, Proteus mirabilis, and Escherichia coli. Peripheral blood lymphocyte reactivity against a number of bacteria was investigated by interferon γ ELISPOT assays. RESULTS—Twins suffering from AS showed cellula…

Biostatistical basis of individualization and segregation analysis using the multilocus DNA probe MZ 1.3: results of a collaborative study.

A collaborative study using the multilocus minisatellite DNA probe MZ 1.3 was carried out to investigate segregation information, mutation rate, DNA fragment frequencies as well as band sharing characteristics. The fingerprint patterns of 393 children as well as 694 unrelated individuals were analysed after digestion of DNA with the restriction enzyme HinfI. A mutation rate of 1% per meiosis or 0.04% per band was found with a mean number of 26 bands/individual. It was shown that maternal and paternal fragments are inherited in equal proportions. Population frequencies of restriction fragments demonstrated a distribution with increasing frequencies in the small fragment size range below 10 k…

C4 DNA RFLP reference typing report.

One hundred and three individual DNA samples (including 23 families) were studied at the gene level during the reference typing of the fourth component of human complement at the VIth Complement Genetics Workshop in Mainz (1989). All samples were analyzed with the restriction enzyme Taq I and with two DNA probes recognizing the 5' ends of both C4 genes and the two adjacent 21-hydroxylase genes. This RFLP is informative for the number of C4 genes as well as for their respective gene size. We found a high degree of variation regarding the number of C4 genes, i.e. haplotypes with 1-3 structural C4 genes of 16 or 22 kb size. By correlating these haplotypes to the complotypes obtained by protein…

Mixture analysis using SWaP™ SNPs and non-biallelic SNPs

Abstract Improved analysis of degraded samples, increased throughput, and a wider choice of typing platforms are some of the significant advantages offered by single nucleotide polymorphism (SNP) genotyping over established short tandem repeat (STR)-based systems. However, DNA mixtures present a considerable problem to SNP analysis as there is currently no generally accepted technique that allows recognition of the presence of a mixed profile or identification of the individual contributors. We present the first demonstration of SNP mixture analysis with an approach based upon the use of two rare subsets of SNPs: SWaP™ SNPs and non-biallelic SNPs and discuss their value for forensic mixture…

Complement genetics: biological implications of polymorphisms and deficiencies

Abstract Complement (C) proteins form a highly complex and important humoral host immune defence system. A recent meeting**The VII Complement Genetics Workshop and Conference was held at Mainz, Germany, on 21–23 May 1998. addressed the role of genetic studies of C components and its regulators with respect to evolution, function and human disease.

Report on the second EDNAP collaborative STR exercise

This report describes an inter-laboratory exercise completed on behalf of the European DNA Profiling (EDNAP) group. The exercise is one in a series designed to identify STR loci which could be used for harmonisation between participating European forensic science laboratories. Participants were asked to identify the alleles present in five bloodstains at the STR loci HUMTHO1 and HUMVWFA31/A. Two of the stains were prepared from mixtures of two different blood samples. There were no special instructions and each laboratory was requested to use the methodology normally employed for crime case investigations. All participating laboratories achieved the same results for both loci. In addition, …

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

Abstract 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 s…

A report of an international collaborative experiment to demonstrate the uniformity obtainable using DNA profiling techniques

This paper describes a collaborative exercise intended to demonstrate whether uniformity of DNA profile results could be achieved between different European laboratories. It was shown that this goal can be obtained provided that a common protocol is followed (specifically the use of a common electrophoretic buffer as being the most important parameter). Generally, lower molecular weight loci (with lower molecular weight fragments) such as YNH24 perform better than higher molecular weight loci such as MS43a. The results of the exercise are discussed in relation to the objectives of the European DNA profiling group (EDNAP).

A brief history of the formation of DNA databases in forensic science within Europe.

The introduction of DNA analysis to forensic science brought with it a number of choices for analysis, not all of which were compatible. As laboratories throughout Europe were eager to use the new technology different systems became routine in different laboratories and consequently, there was no basis for the exchange of results. A period of co-operation then started in which a nucleus of forensic scientists agreed on an uniform system. This collaboration spread to incorporate most of the established forensic science laboratories in Europe and continued through two major changes in the technology. At each step agreement was reached on which systems to use. From the beginning it was realise…

MHC class II genes influence the susceptibility to chronic active hepatitis C

Chronic hepatitis C develops in more than 70% of hepatitis C virus infected subjects. Viral factors influence the disease course, but little is known about the importance of host factors.Frequencies of major histocompatibility complex (MHC) class I and class II antigens were analyzed in two groups of patients with chronic hepatitis C virus infection and in control subjects. MHC class I typing was done by standard microlymphocytotoxicity assays. DRB1 and DQA1 genotyping was done by PCR based typing methods.DRB1*0301 was found in 26 of 75 patients with chronic hepatitis C virus infection (34.7%) and in 12 of 101 control subjects (11.9%) (relative risk 3.9; p0.001). Homozygosity for this allel…

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 seq…

Whole genome amplification—the solution for a common problem in forensic casework?

Abstract To assess the quality of amplified DNA obtained by whole genome amplification, 17 independent STR loci have been typed using two multiplex kits. Results have been compared for correct genotypes, heterozygous peak balance and allelic dropout.

Comparative analysis of short tandem repeats and single nucleotide polymorphisms on the Y-chromosome in Germans, Chinese and Thais.

We have typed genomic DNA samples from 95 individuals from Western Germany, 78 individuals from Bangkok/Thailand and 56 individuals from Chengdu/China for 11 Y-chromosomal diallelic polymorphisms and eight short tandem repeat (STR) systems. For single nucleotide polymorphism (SNP) analysis, a rapid method was applied using the single base extension technology (minisequencing) in combination with capillary electrophoresis. PCR products for SRY-8299, Tat, SRY2627, 92R7, SRY1532, M9, M13, M17/M19 and M20 were pooled and used as templates for the commercially available SNaPshot kit. In addition to these ten SNPs we also tested the Y-chromosomal diallelic Alu repeat insertion DYS287 (YAP) by aga…

Possible association of sudden infant death with partial complement C4 deficiency revealed by post-mortem DNA typing of HLA class II and III genes

Based on evidence of an increased rate of respiratory infections in sudden infant death (SID) infants as well as the observation of familial occurrence, we analysed in a retrospective study class II and class II genes of the major histocompatibility complex in 40 cases of SID by Southern blot analysis of DNA obtained post mortem from tissue samples. In 24 cases, the parents were interviewed and confirmatory human lymphocyte antigen (HLA) and DNA typing was carried out. Using HLA-DR beta and -DQ beta probes, no evidence of an abnormal HLA-DR frequency distribution in SID infants was detected (P = 0.97). Using DNA probes for the tandemly arranged complement C4 and steroid 21-hydroxylase genes…

A functional polymorphism in theIL-10 promoter influences the response after vaccination with HBsAg and hepatitis A

The immune response to hepatitis B surface antigen (HBsAg) is mostly genetically determined. Interleukin 10 (IL-10) is a central immunoregulatory cytokine with important effects on B-cells. We have studied the influence of IL-10 promoter polymorphisms on the immune response to HBsAg and hepatitis A vaccination. We vaccinated 202 twin pairs in an open prospective study with a combined recombinant HBsAg/inactivated hepatitis A vaccine. IL-10 promoter polymorphisms were investigated in all individuals and their influence on anti-HBs, and anti-HAV responsiveness was studied. In the multiple regression analysis accounting for smoking, gender, body mass index and age, the ACC haplotype (-1082, -8…

Association of different tumor necrosis factor α promoter allele frequencies with ankylosing spondylitis in HLA-B27 positive individuals

OBJECTIVE To investigate the potential association of tumor necrosis factor alpha (TNFalpha) promoter alleles with ankylosing spondylitis. METHODS DNA from 141 HLA-B27 positive Caucasian patients with ankylosing spondylitis and 46 B27-positive and 99 B27-negative healthy Caucasian controls was investigated by polymerase chain reaction amplification of the TNFalpha promoter region and subsequent dot-blot analysis with allele-specific oligonucleotides. RESULTS There was a significant decrease in the promoter alleles TNF-238.2 and TNF-308.2 in the ankylosing spondylitis group (266 wild-type alleles, 16 variant alleles) compared with the B27-positive (75 wildtype promoter alleles, 17 variant al…

No Association between Mannose-Binding Lectin Alleles and Susceptibility to Chronic Hepatitis B Virus Infection in German Patients

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 significantl…

Quantification of fluorescent STR genotyping results for chimerism control after bone marrow transplantation

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 de…

Enzyme polymorphisms and haemoglobin variants in Greeks

Several enzyme polymorphisms and hemoglobin variants were typed in a sample of n = 219 non-related Greek blood-donors. The following gene frequencies were observed: pa = 0.201, pb = 0.701, pc = 0.098;PGDA = 0.985, PGDc = 0.015; AK1 = 0.942, AK2 = 0.058; HbA = 0.988, HbS = 0.012. No polymorphic variation was seen in LDH, s-MDH, PHI, or SOD. The population genetical aspects of these results are discussed.

Expression of the human complement C8 subunits is independently regulated by interleukin 1β, interleukin 6, and interferon γ

The eighth component of human complement (C8) is composed of two subunits which are products from three separate genes. The alpha-gamma- and the beta-subunit of C8 are expressed independently, and are part of the membrane attack complex. C8 is primarily synthesized in the liver. It has been shown in previous studies that the human hepatoma cell line HepG2 constitutively expresses C8, and thus is a suitable model system for studying C8 biosynthesis in vitro. Expression is modulated by the cytokines IL-1 beta, IL-6 and IFN-gamma. The effect of the different cytokines on the expression of these subunits was examined using biosynthetical labelling and immunoprecipitation methods. C8 alpha-gamma…

The human complement component C8B gene: structure and phylogenetic relationship

The eighth component of human complement (C8) is a serum protein that consists of three chains (alpha, beta and gamma), encoded by three separate genes, viz., C8A, C8B, and C8G. In serum, the beta-subunit is non-covalently bound to the disulfide-linked alpha-gamma subunit. Using a full-length C8 beta cDNA probe, we isolated several clones from human genomic lambda DNA libraries. Four lambda clones covering the complete cDNA sequence were characterized by TaqI restriction mapping and were "shotgun" subcloned into M13. C8 beta-cDNA-positive clones were partially sequenced to characterize the 12 exons of the gene with sizes from 69 to 347 bp. All intron-exon junctions followed the GT-AG rule. …

STR genotyping and mtDNA sequencing of latent fingerprint on paper

A systematic study was conducted to investigate whether DNA can be successfully extracted from latent fingerprints deposited on ordinary paper and analysed using short tandem repeat profiling and mitochondrial DNA sequencing. In order to evaluate the performance of latent fingerprint analysis in a criminal case, experiments with varying conditions were carried out to improve our understanding of low copy number (LCN) DNA typing. After optimising the extraction methods to achieve increased sensitivity, the examination of touched paper can routinely yield the STR profile of the individual who has touched it. A fingerprint can therefore be considered as a potential source of DNA for genetic id…

DNA polymorphism of the human complement C8 beta gene: formal genetics and intragenic localization.

The eighth component of human complement consists of three subunits of different molecular mass, which are coded for by three separate genetic loci. Polymorphisms have been described at the protein level for the alpha and beta subunits by means of sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Using a full-length human C8 beta cDNA probe, we have studied more than 100 individuals by Southern blot analysis to detect DNA polymorphisms. We have found two restriction fragment length polymorphisms (RFLPs) with the enzymes Taq I and Bam HI. The Taq I polymorphism is defined by two alleles, i.e., a single 4.9 kb fragment or two 2.8/2.1 kb fragments. The allele frequencies are…

Criminal DNA databases: the European situation

In the last 5 years, a number of European countries have successfully introduced national databases holding the DNA profiles from suspected and convicted criminal offenders as well as from biological stain materials from unsolved crime cases. At present, DNA databases are fully or partially in operation in the UK, The Netherlands, Austria, Germany, Finland, Norway, Denmark, Switzerland and Sweden. Furthermore, in the other European countries, specific legislation will be enacted soon, or the introduction of such databases is being discussed to initiate a legislative process. Numerous differences exist regarding the criteria for a criminal offender to be included in the database, the storage…

Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots.

A new STR typing strategy has been developed allowing the simultaneous amplification and subsequent analysis of 11 polymorphic systems with amplicon sizes smaller than 270 bp. The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes by using biotin labelled primers only for one set. Using streptavidin-coated Sepharose beads five STR system…

The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives.

This paper presents an overview of the organisation and the results of the collaborative exercises (CE) of the European DNA Profiling (EDNAP) Group's mitochondrial DNA population database project (EMPOP). The aim of the collaborative exercises was to determine whether uniformity of mtDNA sequencing results could be achieved among different laboratories. These were asked to sequence either the complete mtDNA control region or the two hypervariable regions HVI (16024-16365) and HVII (73-340) from DNA extracts, buccal swabs or bloodstains, proceeding in accordance with the protocol and strategies used in each individual laboratory. The results of the collaborative exercises were employed to id…

Report of a european collaborative exercise comparing DNA typing results using a single locus VNTR probe

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently…

Differential genetic determination of immune responsiveness to hepatitis B surface antigen and to hepatitis A virus: a vaccination study in twins.

Summary Background The course of viral hepatitis is thought to be affected by genetic host variability and, in particular, by genes of the major histocompatibility locus. Hepatitis A and B vaccination is a useful model to study the effect of host factors on the immune response to viral antigens. We aimed to assess the heritability of the HBsAg (anti-HBs) and anti-hepatitis A virus (anti-HAV) immune response and to estimate the effect of the HLA-DRB1 locus and other genetic loci unlinked to HLA. Methods We did an open prospective study and vaccinated 202 twin pairs with a combined recombinant HBsAg/inactivated hepatitis A vaccine. We measured antibodies to HBsAg and HAV and determined HLA-DR…

DNA Commission of the International Society of Forensic Genetics: recommendations on forensic analysis using Y-chromosome short tandem repeats

Abstract During the past few years the DNA commission of the International Society of Forensic Genetics has published a series of documents providing guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. This latest report addresses a relatively new area, namely Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). This report addresses nomenclature, use of allelic ladders, population genetics and reporting methods.

Likelihood Calculations in Paternity Testing on the Basis of DNA-Fingerprints

DNA-fingerprints seem to be a powerful tool in paternity testing. But the quantification of the results in terms of likelihood and likelihood ratios is a problem, because in most cases the correct genetic model and its parameters are not known. Two approaches have been suggested to circumvent these problems. The use of band sharing rates to distinguish between pairs of relatives and pairs of unrelated individuals, and the calculation of likelihood ratios on the basis of simplifying assumptions. The first approach reduces the available genetic evidence to “phenotypic” similarities. The second one makes unjustified simplifying assumptions. These two decision strategies have to be examined wit…

Molecular, genetic, and functional analysis of homozygous C8 beta-chain deficiency in two siblings.

Abstract C8 deficiency is associated with an increased susceptibility to neisserial infections. We present a case of an 11 year old boy who suffered from infection with Neisseria meningitidis . Medical history of the patient and his family ( n = 5) did not indicate any previous immunodeficiency symptoms. Results from the analysis of phagocyte and lymphocyte functions were within the normal range. No hemolytic activities of the classical (CH50) and the alternative (APH50) pathways of complement were measurable, and SC5b-9 protein complexes could not be detected in the patient's plasma. Further analysis by highly sensitive ELISA and functional assays revealed a complete deficiency of C8. Upon…

Recovery of High-Molecular-Weight DNA from Blood and Forensic Specimens

DNA Commission of the International Society of Forensic Genetics: recommendations on forensic analysis using Y-chromosome STRs

During the past few years, the DNA Commission of the International Society of Forensic Genetics has published a series of documents providing guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. This latest report addresses a relatively new area - namely, Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). This report addresses nomenclature, use of allelic ladders, population genetics and reporting methods.

Combined complete C5 and partial C4 deficiency in humans: clinical consequences and complement-mediated functions in vitro.

A family is described with two siblings who suffered at different times from a single episode of meningococcal meningitis by Neisseria meningitidis groups B and C, respectively. In the two subjects, hemolytically active fifth component of complement (C5) was not detectable and antigenic C5 was less than 0.05% and less than 0.7% of normal, respectively. Repletion of sera by purified human C5 (70 micrograms/ml) restored total complement hemolytic activities. The asymptomatic first degree family members had C5 levels compatible with a heterozygous state of C5 deficiency. C4 allotyping revealed an inherited partial deficiency (Q0) of C4A and C4B in the family with a combined C4AQ0 and C4BQ0 het…

Basic issues in forensic DNA typing

DNA analysis has become the standard method in forensic stain typing (termed DNA profiling). In contrast to conventional serological methods, any human tissue or body fluid can be analysed by DNA profiling as long as it contains nucleated cells. The majority of genetic systems studied at the DNA level are derived from "non-coding" portions from the human genome, and are located either in the vicinity of expressed (coding) genes or in stretches of DNA sequences interspersing with the genes. The typing results are usually recorded as DNA fragment lengths or "alleles" indicating the number of core repeat elements for short tandem repeat systems. These typing results do not contain any useful i…

A new polymorphism in the human HFE gene

Failure of sustained engraftment after non-myeloablative conditioning with low-dose TBI and T cell-reduced allogeneic peripheral stem cell transplantation

We investigated whether a T cell-reduced allogeneic stem cell transplant (SCT) with minimal conditioning and subsequent donor lymphocyte infusions (DLI) could reduce the incidence and severity of GVHD while retaining stable engraftment. Five patients with hematological malignancies (three MM, one CLL, one Chediak-Higashi syndrome) were conditioned with TBI (200 cGy). One patient additionally received fludarabine (120 mg/m(2)). CsA and mofetyl-mycophenolate (MMF) were administered to prevent GVHD. All patients were grafted with >3 x 10(6)/kg highly purified CD34(+) cells together with 2 x 10(6)/kg CD3(+) cells (three patients) or 1 x 10(5)/kg CD3(+) cells (two patients). Quick hematopoietic …

A sensitive issue: Pyrosequencing as a valuable forensic SNP typing platform

Analysing minute amounts of DNA is a routine challenge in forensics in part due to the poor sensitivity of an instrument and its inability to detect results from forensic samples. In this study, the sensitivity of the Pyrosequencing method is investigated using varying concentrations of DNA and five autosomal single nucleotide polymorphisms in singleplex on both available instrument models; the PSQ™ 96MA and PSQ™ HS 96A. A detailed comparison of the two models was completed while establishing a lower limit of detection on both instruments to give results supporting the use of Pyrosequencing as a valuable forensic SNP typing platform. © 2005 Elsevier B.V. All rights reserved.

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 isot…

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA ‘fingerprinting’ analysis

Abstract 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 examp…

No primary association between LMP2 polymorphisms and extraspinal manifestations in spondyloarthropathies

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 …

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

Short tandem repeat systems on the X chromosome are the natural counterpart to the well-established Y-chromosomal STR loci. The X-linked systems are inherited as a single haplotype only in males, whereas in females, the X chromosomes recombine and exhibit the same characteristics as the autosomes. Nevertheless, X-linked systems may provide a useful tool in paternity cases with female offspring, in particular when the alleged father is not available for testing, or in forensic identification cases based on the comparison with firstor second-degree relatives. Only a small number of STR loci have been described on the X chromosome, and a number of these are not highly informative. Therefore, w…

Sequence analysis of the DRB1 promoter reveals limited polymorphism with no influence on gene expression.

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 DRB…

Experience with the PCR-based HLA-DQ? DNA typing system in routine forensic casework

The results of HLA-DQ alpha typing from 42 routine forensic cases using the polymerase chain reaction (PCR) were analyzed regarding the reliability, discrimination efficiency and informative value of this system in a given case. The cases included stain typing from a variety of different substates, i.e. blood and semen stains, mixed body fluids, single hairs, cigarette butts, material from fingernail scratches, as well as identification and paternity cases on postmortem and fixed tissue. A total of 125 individual stain and tissue samples were included. PCR amplification was achieved in 70% of these samples. In cases with mixed body fluids, e.g. sperm and vaginal cells from rape cases, DQ al…

Mitochondrial DNA in the central european population

Sequencing of mtDNA is an advanced method for the individualisation of traces. Disadvantages of this method are expensive and time-consuming analysis and evaluation procedures as well as the necessary stock of population-genetic data which is still insufficient. Central European institutes of forensic medicine from Germany, Austria, and Switzerland have been working together since the beginning of 1998 to establish a mtDNA database. The aim is to build up a large stock of forensically established data and provide population-genetic data for frequency investigations, which will serve as a basis for expert opinions and scientific research. Good data quality is ensured by using original sequen…

DNA Fingerprinting as a Tool of Paternity Testing in Germany

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, s…

Sex Determination by Genomic Dot Blot Hybridization and HLA DQα Typing by PCR from Fixed Tissues

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.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.

Abstract A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group [Forensic Sci. Int. 97 (1998) 165]. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a to…

Restriction fragment polymorphisms of the HLA-DR, HLA-DQ, and insulin gene regions in IDDM: The GAW5 data

The primary aim of the insulin-dependent diabetes mellitus (IDDM) component of Genetic Analysis Workshop 5 (GAW5) was to collect and analyze new data on DNA polymorphisms closely linked to the HLA-D region and the insulin gene. The probes and restriction enzymes described here were used by all ten participating labs, and the data from Southern blotting were interpreted and reported according to conventions developed for the Workshop. These DNA data on members of 94 families with two or more IDDM sibs constitute the largest such sample available. The data were used in most of the analyses presented at the Workshop meeting, and are available on request.

Genetic analysis of IDDM: The GAW5 multiplex family dataset

In a collaborative effort by 12 centers from Europe and North America, data were assembled from 94 multiplex families with insulin-dependent diabetes mellitus (IDDM) for analysis of genetic and other factors of possible etiological importance. The dataset contains information on the following genetic markers: HLA-DR beta and -DQ beta restriction fragment length polymorphisms (RFLPs), three RFLPs detected with two probes that map 5' to the insulin gene, the serologically defined HLA loci, and the immunoglobulin allotypes. Data also were included for auto-antibodies to insulin and pancreatic islet cells as possible indicators of pathogenesis and for antibodies to certain viruses that have bee…

Legal aspects of forensic DNA analysis in Germany

DNA analysis may be carried out in cases of civil law (e.g., in cases of disputed paternity) as well as of criminal law (e.g., a case of murder). This overview will concentrate on DNA analysis in the context of criminal law. DNA typing of forensic stain samples is generally carried out in laboratories of the Police and of University Institutes of Legal Medicine. The police laboratories are usually part of the State Criminal Offices (Landeskriminalamt, LKA) or of the central Federal Criminal Office (Bundeskriminalamt, BKA). Each of the sixteen German Federal States (except one) has a LKA with a department carrying out forensic stain analyses. The 25 Institutes of Legal Medicine belong to the…