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.

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.

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.

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.

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.

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.

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.

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.

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.

Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers.

UNLABELLED: In recent years, a large number of groups studied the fate of human stem cells in livers of immunodeficient animals. However, the interpretation of the results is quite controversial. We transplanted 4 different types of human extrahepatic precursor cells (derived from cord blood, monocytes, bone marrow, and pancreas) into livers of nonobese diabetic/severe combined immunodeficiency mice. Human hepatocytes were used as positive controls. Tracking of the transplanted human cells could be achieved by in situ hybridization with alu probes. Cells with alu-positive nuclei stained positive for human albumin and glycogen. Both markers were negative before transplantation. However, cells with alu-positive nuclei did not show a hepatocyte-like morphology and did not express cytochrome P450 3A4, and this suggests that these cells represent a mixed cell type possibly resulting from partial transdifferentiation. Using antibodies specific for human albumin, we also observed a second human albumin-positive cell type that could be clearly distinguished from the previously described cells by its hepatocyte-like morphology. Surprisingly, these cells had a mouse and not a human nucleus which is explained by transdifferentiation of human cells. Although it has not yet been formally proven, we suggest horizontal gene transfer as a likely mechanism, especially because we observed small fragments of human nuclei in mouse cells that originated from deteriorating transplanted cells. Qualitatively similar results were obtained with all 4 human precursor cell types through different routes of administration with and without the induction of liver damage. CONCLUSION: We observed evidence not for transdifferentiation but instead for a complex situation including partial differentiation and possibly horizontal gene transfer.

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.

Compound heterozygous mutations of the SBDS gene in a patient with Shwachman-Diamond syndrome, type 1 diabetes mellitus and osteoporosis.

Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, skeletal abnormalities and hematological dysfunction. The genetic analysis of the SBDS gene and the long-term follow-up of a 37-year-old man with SDS, osteoporosis and type 1 diabetes are reported. Analysis of the SBDS gene revealed a compound heterozygous genotype with 7 mutations. This genotype is the result of the inheritance of abnormal alleles from both healthy parents. We identified putatively non-functional gene conversions from the SBDS pseudogene into the otherwise normal SBDS gene in each of the parentally inherited alleles. The association of SDS and type 1 diabetes mellitus seems to be coincidental and not associated to distinct mutations of the SBDS gene. Osteoporosis in patients with SDS may be the result of a primary defect of the bone metabolism and not of a nutritional problem, although our patient had chronic hypophosphatemia. The long-term follow-up of this patient provides interesting insights into the course of SDS, showing the complexity of genotype-phenotype correlations and the possible influence of other modifying genes and/or environmental factors that might determine the phenotypic presentation of SDS in an individual patient.