German population data for 18 X-STRs: a hexaplex PCR adding two clusters of X-STRs to the Argus X-12 set and expanding the German haplotype databases.

In kinship analysis, large data sets with estimated haplotype frequencies for marker clusters are very important for the likelihood calculation. Practical use of the X-STRs demonstrated that in some complex kinship cases, the marker set of the Investigator Argus X-12 kit can be insufficient. This study aimed to extend the German data base of the Argus X-12 kit (1037 haplotypes) and for a cluster in Xq21 (806 haplotypes) with additional 700 male haplotypes and to include a further cluster in Xp22.3 to complete the X-STR marker set for complex kinship cases.

X-chromosomal 21-indel marker panel in German and Baltic populations.

In order to verify specific biallelic X-indels and their characteristic properties in distinct populations, one German and three Baltic population groups (Estonia, Latvia, and Lithuania) have been analyzed by a short amplicon method, which also enables detection of degraded DNA samples. To combine 21 indels in a single multiplex PCR, all products were arranged according to their expected amplicon length (~40-160 bp) on the basis of three different fluorochromes. Separation of PCR products was carried out in a single capillary electrophoresis. Data evaluating was performed including five further indel markers which have already been tested in identical samples, resulting in altogether 26 markers. The majority of the genetic material showed combinations of insertion elements (L-fragments). Combinations of deletion elements (S-fragments) in contrast occurred with significant lower ratios. Hardy-Weinberg equilibrium (HWE) was observed for all markers except for MID1361 and MID329. This was attributed to an insufficient number of samples. For two known linkage groups within the 26-indel set (MID357-MID356 and MID3690-MID3719-MID2089), haplotype data were determined. A pairwise comparison of German and Baltic allele frequencies did not show significant deviation. This result indicates a possible genetic association between all four population groups. The calculated biostatistical parameters show high forensic efficiency for this set of indel markers. In a segregation analysis investigating 194 meiosis, no mutations have been detected regarding expected transmission patterns.

Long QT syndrome mutation detection by SNaPshot technique.

Long QT syndrome (LQTS) is a cardiac disorder with an abnormality of cardiac rhythm associated with sudden death especially in younger, apparently healthy individuals. If there is no clear cause of death detectable during comprehensive coroner's inquest (autopsy-negative cases), you have to consider LQTS and other heritable arrhythmia syndromes. A molecular genetic screening regarding mutations in associated genes can help to ensure the cause of death and to protect affected family members. Genetic testing of LQTS, currently performed mainly by sequencing, is still very expensive and time consuming. With this study we present a rapid and reasonable method for the simultaneously screening of some of the most common mutations associated with LQTS, focused on the KCNQ1 and KCNH2 genes. With the method of SNaPshot minisequencing, a total of 58 mutations were analyzed in four multiplex assays which were successfully established and optimized. The comparison with samples previously analyzed by direct sequencing showed concordance. Furthermore, autopsy-negative cases were tested but no mutations could be observed in any of the specimen. The presented method is well suitable for LQTS mutation screening. An enhancement to further mutations and population-based investigations regarding mutation frequencies should be the aim of prospective studies.

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.

Adenoid squamous carcinoma (pseudoangiosarcomatous carcinoma) of the vulva: a rare but highly aggressive variant of squamous cell carcinoma-report of a case and review of the literature.

Pseudoangiosarcomatous squamous cell carcinoma is an unusual but aggressive variant of acantholytic squamous cell carcinoma of the vulva that mimics angiosarcoma on histology. We present a case of a 57-year-old woman with bilateral inguinal metastatic disease at the time of diagnosis, who died 4 months later because of distant metastatic disease to the lungs. Molecular analysis did not reveal any human papillomavirus infection. Because of the positive p53 immunostaining and the association to lichen sclerosus and simple type of high-grade vulvar intraepithelial neoplasia, alteration of p53 tumor suppressor gene might be involved in the pathogenesis of vulvar pseudoangiosarcomatous squamous cell carcinoma. However, further molecular studies are required.

Spontaneous remission of acute myeloid leukemia relapse after hematopoietic cell transplantation in a high-risk patient with 11q23/MLL abnormality.

A 35-year-old female patient was diagnosed with acute myeloid leukemia with multiple genetic aberrations [48 XX, del(3)(q21), +6, t(11;15)(q23;q15), +21] including an 11q23/MLL abnormality. The patient achieved a complete remission after one induction chemotherapy cycle. After three courses of consolidation, a matched unrelated hematopoietic cell transplantation (HCT) was performed. Following an upper respiratory tract infection 7 years after transplant, her blood counts declined to leukocytes of 1 x 10(9)/l, platelets of 51 x 10(9)/l and hemoglobin of 7.5 g/dl. A bone marrow aspirate revealed 55% leukemic blasts carrying the unfavorable genetic aberrations seen at initial diagnosis (11q23/MLL). In the absence of any disease-specific treatment, the leukemic blasts cleared from the bone marrow within 6 days after diagnosis of relapse and peripheral blood counts returned to normal. Molecular analysis of the 11q23/MLL rearrangement was used to evaluate minimal residual disease, which became undetectable in repetitive FISH analyses. This is the first report of spontaneous remission in a patient with initially a multiaberrant leukemic cell clone and a proven 11q23/MLL abnormality at relapse after HCT.

Population data of Y-chromosomal STRs in Russian males of the Primorye region population.

Data of eight Y-chromosomal STRs, the so called "minimal core set", were obtained from 152 unrelated males of the Primorye region of Russia. The allelic frequencies correspond to other European populations. The background is a settlement of males from the European part of Russia, Ukraine and other states which were included in the former western part of the Soviet Union. On the other hand the distribution of the most frequent haplotypes differs to the Ukraine and Russian population. The most frequent haplotype was obtained five times in the population corresponding to 3.3%. The haplotype data reported here have been included into the Y-STR database maintained at the Institute of Legal Medicine, Humboldt-University, Berlin.

Validation of the X-linked STR DXS6801.

This paper presents sequence and population genetic data of the X-linked microsatellite marker DXS6801 (GDB:G00-365-276) which is a tetranucleotide repeat polymorphism representing eight alleles 109-141 bp in length. Data were obtained from a sample of 1146 unrelated German individuals. DXS6801 is located 99.7 cM from Xptel, nearby DXS6809 [Int. J. Legal Med. 117 (2003) 241] and DXS6789 [Forensic Sci. Int. 119 (2001) 42] in Xq21. The new marker could be added to the panel of ChrX STRs, especially usable as a part of the Xq21 linkage group.

The X-linked STRs DXS7130 and DXS6803.

This paper presents sequence and population genetic data of two new X-linked microsatellite markers, suitable for forensic purposes. Data were obtained from a sample of unrelated German individuals (male and female). Two highly informative markers could be added to the panel of ChrX STRs [J. Edelmann, S. Hering, M. Michael, R. Lessig, D. Deichsel, G. Meier-Sundhausen, L. Roewer, I. Plate, R. Szibor, 16 X-chromosome STR loci frequency data from a German population, For. Sci. Int. 124 (2001) 215-218; J. Edelmann, D. Deichsel, S. Hering, I. Plate, R. Szibor, Sequence variation and allele nomenclature for the X-linked STRs DXS9895, DXS8378, DXS7132, DXS6800, DXS7133, GATA172D05, DXS7423 and DXS8377, For. Sci. Int. 129 (2002) 99-103].

Validation of the STR DXS7424 and the linkage situation on the X-chromosome.

X-linked microsatellite markers have proven to be powerful tools for parentage testing, mainly in deficiency paternity cases when the disputed child is female. However, only a small number of X-linked short tandem repeats (STRs) have been comprehensively described for forensic applications to date. We present sequence and population genetic data of the DXS7424 STR (GDB-G00-577-633) which is a trinucleotide repeat polymorphism representing 12 alleles of 147-180 bp in length. DXS7424 is located at Xq22 and closely linked to DXS101, corresponding to a genetic localisation of 104.9-121 cM from Xp-tel.PCR fragment length measurements and sequencing were carried out using the automatic gene analyser ABI 310 (Applied Biosystems). The population of 764 unrelated Germans checked for this STR exhibited the following features: polymorphism information content (PIC) = 0.780; heterozygosity (Het) = 0.843; mean exclusion chance (MEC = 0.766. Kinship tests revealed a typical X-linked inheritance. In 300 meioses under investigation, mutations were not found. Significant deviations from the Hardy-Weinberg equilibrium (HWE) were not established. Linkage studies confirmed closely linkage to DXS101. Additional we found linkage disequilibrium between DXS7424 and DXS101. This requires to use the established haplotype frequencies in kinship testing.

Sequence variation and allele nomenclature for the X-linked STRs DXS9895, DXS8378, DXS7132, DXS6800, DXS7133, GATA172D05, DXS7423 and DXS8377.

X-linked DNA markers are increasingly used in forensic kinship testing. This paper presents sequencing data of the short tandem repeats (STRs) DXS9895, DXS8378, DXS7132, DXS6800, DXS7133, GATA172D05, DXS7423, DXS8377 and proposes an allele nomenclature. Alleles were assigned according to the recommendations of the International Society of Forensic Genetics (ISFG) Commission.

Cell line DNA typing in forensic genetics--the necessity of reliable standards.

The incorporation of reference DNA is crucial to the validation of any DNA typing protocol. This paper aims to provide a panel of reference DNAs for actual forensic profiling strategies, i.e. autosomal and gonosomal STR typing as well as mtDNA sequencing. We have characterised three human lymphoid cell lines, GM9947, GM9948 and GM3657, and considered 58 autosomal and gonosomal microsatellites as well as the mitochondrial control region sequence. Well-established markers and STRs recently developed for forensic use were involved. K562 DNA samples which we purchased from two different suppliers were also analysed. They revealed conflicting results with regard to the ChrX STR marker genotype. Hence, we suggest that K562 is no longer used for the calibration of profiling techniques. Our investigation establishes a panel of one female and two male DNA samples as an STR allelic ladder calibration tool and offers information on six alleles of each autosome (AS) marker, three alleles of each X chromosome (ChrX) marker and two alleles of each ChrY marker. In addition, sequences of the mitochondrial control region of the three DNAs are communicated in order to provide sequencing quality control.

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.

The Y-chromosomal STRs DYS481, DYS570, DYS576 and DYS643.

The Y-STRs DYS481, DYS570, DYS576, and DYS643 were investigated in a West Saxonian population sample, which have been previously typed with the well known minimal or extended haplotype of Y-STRs. Observed allele frequencies are reported along with the allele nomenclature based on sequencing. Gene diversities as well as the haplotype diversity of the four markers--DYS481, DYS570, DYS576 and DYS643--were calculated and compared with published data of other population samples. The discrimination capacities of the markers show a high potential for forensic purposes.

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.

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.

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.