Sandra Hering

Use of X-linked markers for forensic purposes

In forensic science, X-chromosomal short tandem repeats (ChrX STRs) bear the potential to efficiently complement the analysis of other genetic markers (autosomal, Y-chromosomal or mitochondrial). We review the population genetic properties and forensic utility of selected ChrX markers, and discuss the problems and limitations arising with their practical use. Formulae required to assess the evidential power of individual markers in different contexts are summarised and applied to ChrX STRs of interest. Since linkage and linkage disequilibrium between markers affect the inferential interpretation of genotype data, practically relevant information regarding the co-localisation and haplotypic association of ChrX STRs is provided. Finally, two examples of complex kinship testing are presented which serve to highlight the particular importance of ChrX STRs for solving deficiency cases and cases involving blood relatives.

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.

Radiobiological hypoxia, histological parameters of tumour microenvironment and local tumour control after fractionated irradiation.

OBJECTIVE To investigate the relationships between radiobiological hypoxic fraction (rHF), pimonidazole hypoxic fraction (pHF) as well as other histological parameters of the tumour microenvironment, and local tumour control after fractionated irradiation in human squamous cell carcinomas (hSCCs). MATERIAL AND METHODS Ten different hSCC cell lines were transplanted into nude mice and rHF was calculated from local tumour control rates after single dose irradiation under normal or clamped blood flow conditions. In parallel, tumours were irradiated with 30 fractions within 6 weeks. Radiation response was quantified as dose required to cure 50% of tumours (TCD(50)). Unirradiated tumours were excised for histological evaluation including relative hypoxic area (pHF), relative vascular area (RVA), and fraction of perfused vessels (PF). RESULTS A weak but significant positive correlation between rHF (R(2)=0.6, p=0.014) and TCD(50) after fractionated irradiation was found. The pHF did not correlate with rHF but was significantly associated with the TCD(50) after single dose clamp (R(2)=0.8, p=0.003) and showed a trend for an association with TCD(50) after fractionated irradiation (R(2)=0.4, p=0.067). Relative vascular area and fraction of perfused vessels did not show an association with rHF or TCD(50) after fractionated irradiation. CONCLUSIONS Our data suggest that radiobiological hypoxia contributes to the response after fractionated irradiation but that also other radiobiological mechanisms are involved. In the present study, pimonidazole labelling does not reflect rHF and has a limited value to predict local tumour control after fractionated irradiation. The association between pHF and TCD(50) after single dose clamp warrants further investigation.

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.

CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell lines HCT-116

BACKGROUND AND PURPOSE CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133(+/-) subpopulations of one cell line of interest. MATERIALS AND METHODS Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133+ and CD133(-) HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice. RESULTS AND CONCLUSIONS CRC cell lines could be classified into three groups: (i) CD133(-), (ii) CD133+ and (iii) those with two distinct CD133+ and CD133(-) subpopulations. Isolated CD133(+/-) HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133(-) HCT-116 showed a striking enrichment in the CD133+ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell lines. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.

Development of the X-linked tetrameric microsatellite marker HumDXS6789 for forensic purposes

This paper presents sequence and population genetic data of the X-linked DXS6789 short tandem repeat (STR). The tetranucleotide repeat polymorphism DXS6789, also known as CHLC.GATA31F01, is located at the Xq22.3 region. This locus is unlinked with DXS6807 and slightly linked with ARA, DXS9898 and HPRTB. In kinship testing, DXS6789 is suitable for concomitant use with DXS6807. Population genetic data were obtained by analysing 250 unrelated males and 315 females from East Germany. In this population, the STR exhibited 12 clearly distinguishable alleles ranging from 154 to 198bps in length. DXS6789 is characterised by the following data: polymorphic information content (PIC)=0.70; observed heterozygosity (Het)=0.78; mean exclusion chance (MEC)=0.70. A deviation from the Hardy-Weinberg equilibrium could not be detected. The investigations we performed in 243 mother-child and 161 father-child meioses did not reveal any mutations.

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.

Development of the X-linked tetrameric microsatellite marker DXS9898 for forensic purposes.

HumDXS9898 also known as CHLC x GATA 126G01 is a tetrameric microsatellite marker located at the Xq21.33 pericentromeric region. In kinship testing HumDXS9898 is suitable for concomitant use with HumHPRTB and HumDXS6807 which are separated from HumDXS9898 by genetic map distance of 150 and 80 cM, respectively. HumDXS9898 is closely linked to HumARA. In the German population, HumDXS9898 exhibits seven clearly distinguishable alleles ranging from 189 to 214 basepairs in size. Deviation from Hardy-Weinberg equilibrium could not be detected. The observed heterozygosity was 0.75 for females and the mean exclusion probability was 0.73 for female children. Mutations were not found in the present material.

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.