Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome.

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

[Carbon monoxide poisoning from indoor barbecues--incidents reported to the German-speaking Poison Information Centers and the German Federal Institute for Risk Assessment (BfR) in Berlin]. / Kohlenmonoxidvergiftungen durch Grillen in Innenräumen--Mitteilungen an die deutschsprachigen Giftinformationszentren und das Bundesinstitut für Risikobewertung Berlin.

From 2008 to the end of 2009 the Joint Poison Information Center (PIC) in Erfurt observed 7 incidents involving 17 persons (1 fatality) with signs of carbon monoxide poisoning from indoor barbecues (COFIB). To find out whether COFIB is a regional or a general phenomenon in Germany, Austria and Switzerland, all information about COFIBs recorded by the 11 German-speaking Poison Information Centers and the BfR Berlin were retrospectively analyzed for the period 2000 to 2009. In all, 60 COFIBs (accidental: 90.0 %, suicidal: 8.3%, reason unknown: 1.7%) involving 146 individuals were reported. The number of incidents increased from one case with 2 persons in 2000 to 18 cases involving 34 persons in 2009. The 146 victims (female 26.7%, male 27.4%, gender unknown 45.9%; adults 58.2%, children 24.7%, age unknown 17.1%) lived in 15 of the 16 federal states of Germany and in Switzerland. The highest number of victims was found in Bavaria (23), Brandenburg (18), and Baden-Wuerttemberg (18). The symptoms according to the Poisoning Severity Score were none to mild in 60.3%, moderate in 13.7%, severe in 11.6%, fatal in 6.9% and unratable in 7.5%. No clear correlation was found between the carboxyhemoglobin concentration and the severity of the symptoms. As a rising number of COFIBs often involving several individuals was observed from 2000 to 2009, the general public was informed about the risks of indoor barbecues.

Evaluation of seven X-chromosomal short tandem repeat loci located within the Xq26 region.

In this study a set of 29 X-chromosomal short tandem repeats (STRs) located within the Xq26 region was evaluated. These STRs were found within the 133.14-133.45Mb region around the HPRTB locus. Evaluation of the microsatellites was performed with regard to polymorphism, reliable amplification, and low stutter artefacts. DXS10101, DXS10102, and DXS10103 were identified as those X-STRs with highest diversity; i.e. PIC values of 0.7174-0.8933. The locus DXS10101 was the optimal candidate for the integration in the commercial available test system Mentype Argus X-8 PCR amplification kit.

X chromosomal recombination--a family study analysing 39 STR markers in German three-generation pedigrees.

Typing of polymorphisms on the human chromosome X (ChrX) has become a standard technique in forensic genetics, and a growing number of short tandem repeats (STRs) has been established. Knowledge of marker recombination is of great significance especially when ChrX typing is used in forensic kinship testing. It is known that meiotic recombination is not a simple function of physical distance but crossing over events tend to be clustered. Information on genetic distances between markers can be gathered by family studies and by interpolation of gene bank data such as the Rutgers map. We typed DNA samples of pedigrees consisting of mothers with several sons and grandfather-mother-son constellations and report here the recombination characteristics of 39 ChrX STRs in up to 135 meioses.

Validation of six closely linked STRs located in the chromosome X centromere region.

We propose that clusters of closely linked markers, which segregate as stable haplotypes, provide a high potential to solve complex kinship cases. It is known that the X-chromosomal centromere region shows an extremely low degree of recombination. Hence, we focused our interest on the region between 56 and 64 Mb distant from the Xp telomere and considered 6 STRs which are now registered in the Genome Data Base as DXS10161, DXS10159, DXS10162, DXS10163, DXS10164, and DXS10165. All of these markers show a tetranucleotide or pentanucleotide structure and exhibit high or medium polymorphic information content. As a peculiarity, DXS10163 is a combination of a pentanucleotide STR and an 18 bp INDEL polymorphism. We report here the primer sequences, the repeat structures, the allele distributions and parameters of forensic interest for a German population sample.

Characterisation of the STR markers DXS10146, DXS10134 and DXS10147 located within a 79.1 kb region at Xq28.

Three polymorphic X-chromosomal STR markers within a 79 kb region at Xq28 were studied and registered in the GDB as DXS10146, DXS10134 and DXS10147. These markers were molecular characterised and evaluated for their forensic usage. As a result DXS10134 was recently integrated in the commercial available test kit Mentype Argus X-8. At locus DXS10146 we found 23 alleles with PIC and HET values of 0.878 and 0.887. Locus DXS10134 showed 17 alleles with PIC and HET values of 0.844 and 0.858. At locus DXS10147 only 5 alleles with some lower PIC and HET values of 0.636 and 0.692 were found. Additionally, the already known and closely linked STR DXS7423 was included into the haplotyping and recombination studies. Testing this cluster a German population of 404 males revealed the presence of 311 haplotypes. Recombination analysis was performed in 109 father-daughter-grandson trios in which two crossing over events were observed located in the 65.8 kb region between DXS10146 and DXS10134. By using this STR complex for haplotyping in kinship testing further genetic analyses are required to establish an exact recombination rate.

Population genetic evaluation of eight X-chromosomal short tandem repeat loci using Mentype Argus X-8 PCR amplification kit.

The evaluation of four pairs of X-chromosomal short tandem repeats (STRs), i.e. DXS10135-DXS8378, DXS7132-DXS10074, HPRTB-DXS10101 and DXS7423-DXS10134 was carried out using the Argus X-8 Multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome (ChrX), and for practical reasons they are assigned to four linkage groups 1-4. The genetic distance within the STR pairs is assumed to be <1cM, whereas the pair to pair space is about 50 cM or more. Here, we present single STR allele frequencies, haplotype frequencies of the respective STR pairs and further population genetic parameters of forensic interest. Most data refer to a German population, however small samples from Ghana and Japan were also investigated. Furthermore, sequencing of all STR loci displayed the presence of microvariant alleles and variations in the repeat flanking region. A total of 350 meioses investigated here revealed only one sperm DXS7132 mutation. For analysis of linkages within the STR pairs a study involving 104 female meiosis with respect to recombination events was performed. The STR panel presented here provides a powerful tool for solving complex kinship in the case that X-chromosomal lineages can be taken under investigation.

The STR cluster DXS10148-DXS8378-DXS10135 provides a powerful tool for X-chromosomal haplotyping at Xp22.

The evaluation of four pairs of tightly linked chromosome X (ChrX) short tandem repeat (STR)s at Xp22, Xq12, Xq26 and Xq28 led to the creation of the Argus X 8 multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome, and for practical reasons, they are assigned to four linkage groups 1-4. To achieve a further considerable enhancement in discrimination power, we suggest to include additional markers. A recent paper referred to the earlier evaluation of STR clusters at Xq12, Xq26 and Xq28, and here we present the pending data of linkage group 1 at Xp22. The newly established STR updates the Xp22 STR cluster which now presents three polymorphic markers: DXS10148 (PIC = 0.8556), DXS10135 (PIC = 0.9093) and DXS 8378 (PIC = 0.6454). Typing of 398 X-chromosomes provided 278 different and 200 unique haplotypes. All the other haplotypes observed appeared with frequencies in the range between 0.005 and 0.015. Considering this STR triple in the context with the three further triple clusters Xq12, Xq26 and Xq28 published earlier, we announced the development of a next generation of a ChrX STR cluster typing kit.

Complex variability of intron 40 of the von Willebrand factor (vWF) gene.

Intron 40 of the von Willebrand factor (vWF) gene exhibits a highly variable region of about 0.65 kb, which contains 5 juxtaposed STRs. We sequenced 0.65 kb amplicons from 68 chromosomes and found 2 frequent indel polymorphisms and 5 SNPs. The 68 chromosomes investigated here presented a total of 47 different haplotypes. Regarding the SNP allele distribution in our sample, we arranged our results of the vWF intron 40 into a system of 3 haplotypes, i.e. haplotypes a, b and c. Our review may be valuable in further optimising vWF typing in forensic applications and in avoiding pitfalls. Further attempts to develop sophisticated techniques may soon enable haplotyping using autosomale STR clusters.

X-chromosomal markers: past, present and future.

Experience gained in clinical genetics led to the fundamental idea of using X-chromosomal markers in a wide range of forensic applications. To date more than 30 STRs have been established as forensic markers. Joint typing of very tightly linked STRs yields stable haplotypes, and can be used for establishing the relationship between distant relatives, such as aunt-niece pairs and cousins. For such applications the new ChrX typing kit Argus X-8 which is commercially available now is a powerful tool. This paper is aimed at presenting a brief survey of historical developments and discussing present and future aspects of forensic X-chomosomal testing.

New alleles and mutational events at 14 STR loci from different German populations.

The molecular origin of DNA mutations and the mutation rates were analyzed at 14 short tandem repeat (STR) loci with samples from trio cases derived from 10 different German population samples. STR loci comprised of D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D12S391, D18S51, D19S246, D20S480, D21S226, and D22S689. In a total of 488 meioses, 16 isolated genetic inconsistencies in 8 different STRs were observed, whereas no mutations were found at the other loci. The data of five mutations suggested the presence of silent or null alleles due to sequence variation in primer binding site. This could be confirmed for four suspected cases by the use of alternative primer sets and by DNA sequence analyses. Furthermore, this study revealed nine new allelic variants at five different loci.

Mitochondrial D-loop (CA)n repeat length heteroplasmy: frequency in a German population sample and inheritance studies in two pedigrees.

Sequence analysis of the human mitochondrial genome (mtDNA) has proven to be a valuable tool in forensic identity testing and the analysis of crime scene stains. In contrast to the very expensive sequencing technique, typing of different length variants can greatly facilitate screening of a large number of traces for their relevance during casework. Within the mitochondrial control region, a dinucleotide (CA)( n ) repeat locus is present. To assess the discrimination power of this marker, we have determined (CA)( n ) allele distribution and the frequency of heteroplasmy in a population sample of 2,458 Germans. The inclination to develop heteroplasmic mixtures (CA)( n )/(CA)( n-1) was positively correlated with the number of CA repeats in the mtDNA. In addition, we have studied the inheritance patterns of (CA)( n ) repeat sequence heteroplasmy in two pedigrees. In one pedigree, we also found a length heteroplasmy in the homopolymeric C-tract (nt 303-309). Our data show stable inheritance of heteroplasmy within the homopolymeric C-stretch, but rather unstable inheritance regarding the (CA)( n ) repeat locus.

Forensic mass screening using mtDNA.

At the forensic autopsy of a sexual murder victim, some trace hairs, possibly belonging to the perpetrator, were saved. Initially, the analysis of a pubic hair shaft only revealed the presence of the mitochondrial (mt) DNA haplotype profile consisting of the (CA)(6) allele and the complete hypervariable region 1 (HV1) and 2 (HV2) sequence. Later, typing of some further telogene trace hairs, which had been stored for several years, yielded a nuclear short tandem repeat (STR) profile. We used both the mtDNA haplotype and the STR profile to start a DNA mass screening project involving 2,335 male citizens of the relevant communities. MtDNA screening was carried out by using the CA repeat amplification in combination with an SNP typing procedure based on the restriction site analysis of amplified d-loop sequences. The aim of our paper is to put mass screening with mtDNA up for discussion.

DXS10079, DXS10074 and DXS10075 are STRs located within a 280-kb region of Xq12 and provide stable haplotypes useful for complex kinship cases.

The evaluation of the short tandem repeat (STR) markers DXS10079, DXS10074 and DXS10075 was amended to establish a STR cluster spanning a genetic distance<1 cM. These three STRs are located within a 280-kb region at Xq12 and provide stable haplotypes useful for solving complex kinship cases. Theoretically, this cluster could give rise to 2,548 different haplotypes in the German population and the genotyping of 781 men revealed the presence of 172 haplotypes. Since the three STRs were shown to be in strong linkage disequilibrium (LD), haplotype frequencies cannot be computed on the basis of a single locus allele frequency alone but have to be estimated directly. Here, we present data on linkage, haplotype frequencies and LD in a German population. Further clusters from other regions of the X chromosome will be published in the future to cover the chromosome with a well-structured network of highly informative sites.

DXS6797 contains two STRs which can be easily haplotyped in both sexes.

DXS6797 is a complex X-chromosomal locus which contains two variable short tandem repeats (STRs) (motif ATCT) separated by 128 non-polymorphic nucleotides. The two STRs can be cleaved apart by Taq I digestion. Conventionally, DXS6797 is typed by measuring the overall amplicon length, providing only eight alleles [polymorphism information content (PIC) 0.733, mean exclusion chance (MEC) 0.712]. Separate amplification would increase the discrimination but obscure the haplotype constellation in females. Therefore, we proceed by amplifying the whole sequence containing both repeats (DXS6797 I and DXS6797 II) using a Fam-labelled forward primer and a Tamra-labelled reverse primer. We then measure the length of the entire double-labelled amplicon and a Taq-I-digested aliquot to infer, for both males and females, compound haplotypes consisting of DXS6797 I and DXS6797 II repeat length. This procedure has the potential to provide 42 DXS6797 haplotypes. If the crossover rate between both STRs is assumed to be <1.5x10(-6), DXS6797 haplotypes could be used for kinship testing like STR alleles. In our German sample (780 X chromosomes), we determined 27 haplotypes (PIC 0.842, MEC 0,834) and in 220 meioses, we found no new mutations.

A new Web site compiling forensic chromosome X research is now online.

We would like to announce the opening of a new Web site ( http://www.chrx-str.org ), which contains a database surveying current research on chromosome X markers used for forensic purposes, evolutionary anthropology and other genetic research. Currently, we summarise short tandem repeat data with regard to the physical and genetic localisation, repeat structure, allele nomenclature, mutation rates and population genetics. In the future, we may include diallelic markers. The results contained in this database come from published journal articles. The authors of published articles are invited to complement their own papers by submitting data obtained from follow-up studies here. Furthermore, population data which are not able to find space in journals may be published at this Web site. The growing field of ChrX haplotyping is producing an extensive amount of data, which requires a place that can complement journal publications.

Haplotyping of STR cluster DXS6801-DXS6809-DXS6789 on Xq21 provides a powerful tool for kinship testing.

Short tandem repeat (STR) markers DXS6801 (GATA41B11), DXS6809 (GATA69B129) and DXS6789 (GATA31F01) are located in a 3-Mb region on human chromosome Xq21, spanning approximately 3-6 cM. Theoretically, this cluster could give rise to 1,144 different haplotypes in the German population. In fact, genotyping of 806 males revealed the presence of 207 different haplotypes. Since the three STRs have been shown to be in strong linkage disequilibrium (LD), haplotype frequencies cannot be computed on the basis of single locus allele frequencies alone, but have to be estimated directly instead. In this work, we present data on linkage, haplotype frequencies and LD in the German population. To highlight the potential of the STR cluster for forensic analysis, we also report two examples of its successful application in pedigree-based kinship testing.

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

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