The typing of X-chromosomal STR markers (ChrX STRs) is of great importance in kinship testing, especially in deficiency cases, where there is no available DNA sample from one or both parents. ChrX STRs also have increasing applicability in forensic cases, even those where only very short fragments of DNA can be obtained. Dr. Jeanett Edelmann of the X Working Group is a key researcher in the field of ChrX STR application in kinship and forensic testing.
Dr. Edelmann has been with the Institute of Forensic Medicine of Leipzig University in Germany since 1990. Her major focus is on ChrX STRs in human identification. As one of the founders of the X Working Group, she was a major driving force behind research into ChrX STRs, and she co-created the Forensic ChrX Research website, which has an extensive online database for ChrX STRs. She recently spoke to QIAGEN about her research and some interesting cases where X-chromosomal STR markers were the deciding factors.
Welcome to QIAGEN, Dr. Edelmann. Could you tell us how you came to focus on X-chromosomal STR markers?
After studying biology at the University of Leipzig and working in the field of immunology for a couple of years, I joined the Institute of Forensic Medicine. That was 1990, so my research started in the time of RFLP and VNTR analysis. As analytical methods changed, so of course did the focus of my laboratory. Nowadays, my team uses highly automated STR analysis.
We performed several population studies at the Institute during the nineties, each using the most current DNA sample and assay technology. In the mid-nineties, I started working on projects using our validated STR markers. My team and I successfully performed chimerism studies in leukemia patients following stem cell transplantation, and loss of heterozygosity studies in cases of cervical carcinoma.
Around this time, we started to investigate gonosomal STR markers, initially using Y-chromosomal STR markers (ChrY STRs), which led to a large number of population studies.
Since 1998, I have focused my interest on X-chromosomal STR markers (ChrX STRs). In 1998, ChrX STRs were still unexploited, but today a range of these markers have been characterized and can now be used to solve complex pedigrees.
What are the main tasks of your laboratory?
We have three main tasks. We do a great deal of paternity testing, specializing in solving deficiency cases using gonosomal STRs as an additional marker category. Most of our time is spent on stain analysis for criminal cases. We also perform chimerism analysis on leukaemia patients following stem cell transplantation.
Is your team doing any other forensic research?
We have started a project analysing SNPs in cases of cardiac disorders associated with sudden death. A graduate student of my laboratory is involved, but I’m afraid I can’t say anything more about that project at this time.
Coming back to gonosomal STR markers, you were one of the key researchers in the development in our understanding of ChrX and its application in human identity testing. Could you tell us how that came about, and how the X Working Group developed?
I started to validate ChrX STRs for forensic applications about 13 years ago when I was working on my dissertation. My advisor was Professor Szibor from Magdeburg. At that time, only two markers were applied in forensics: HPRT and ARA. Shortly thereafter, Dr. Sandra Hering from Dresden identified ChrX markers. Then we started to establish a number of new ChrX STRs for forensic use and published a lot of data regarding allele-sequence structure, allele frequencies, and mutation rates. With this research, we were able to establish the ChrX STRs as a new marker category because their genotyping complements the analysis of autosomal and Y-chromosomal markers very efficiently, especially in complex kinship testing.
Since all of the newly identified markers were located on the same chromosome, it became necessary to understand their linkage. Because the physical and genetic localization of markers is not strictly linear on the X-chromosome, marker localization is a particularly important issue in ChrX-based kinship testing. We proceeded with a focus on linkage and linkage-disequilibrium studies and created the X Working Group. We are a small group, but all very dedicated to elucidating this topic. Creating this group allowed us to efficiently coordinate and divide our work in order to finally assemble all our data.
The cooperation with Professor Szibor and Dr. Hering was extremely beneficial in this work, and I am very thankful for it.
What has changed in ChrX-testing technology in this time?
The typing of ChrX markers increasingly became an issue in kinship testing, especially in deficiency cases, where ChrX markers can be used rather than autosomal or ChrY markers. The rising demand for ChrX markers required the development of standardized test kits for the user. This was done in cooperation with the company Biotype Diagnostics, who produced the first X-STR test kit for forensic use. The kit comprised four previously validated unlinked markers. Later, a second X-marker kit was developed, which contained 8 markers. Today QIAGEN’s Investigator Argus X-12 kit is the standard for X-marker testing. The 12 markers of the Investigator Argus X-12 are clustered into 4 linkage groups (3 markers per group), so each set of 3 markers is handled as a haplotype for genotyping. The first haplotype frequency data for the Argus X-12 was recently published.
Since ChrX STRs are only applicable to a minority of cases, the extent of frequency and population data rose slowly. As forensic certainty can only be achieved with a solid base of data, it seemed to be advisable to collect marker data, such as allele distributions and mutation rates, in an online database accessible to the whole forensic community. This is what led us to create our ChrX website in 2005, which today is managed by Qualitype AG, Dresden.
Now, a large number of ChrX STRs are available, and their linkage situation has been verified in extensive recombination studies. The haplotype frequencies of closely linked markers are the basis for actual ChrX STR use in special kinship cases.
What is a potential area of improvement in ChrX marker use?
Understanding the linkage of markers and identifying the haplotypes of linkage groups, which is not usual in autosomal STR analysis, is sometimes not considered. But overall the X Working Group is proud to have created the basis for the application of all of these markers.
Are ChrX STRs applied in all kinship cases?
No, they are not. Ordinary paternity cases do not require any additional or alternative markers. They can usually be solved using autosomal STRs. However, when the relationship between a father and daughter is in question, the inclusion of ChrX markers can be useful because of their higher mean exclusion chances.
Could you share an interesting example where ChrX markers were used to solve a court case?
We performed the identification of a deceased man. The only existing person for alignment was a possible half-brother with the same mother. Brothers share a given maternal X-chromosome allele with a probability of 0.5. We tested about 20 ChrX STRs and found accordance along nearly the whole X-chromosome, from Xp22 to Xq28. Only at the top of Xp could two markers be distinguished, explainable by a crossing-over event.
Have you had any technically challenging cases that were successfully resolved based on recent developments in ChrX STR analysis?
There is an interesting example involving 3 women. We’ll call them R, K, and H. R and K had the same mother (m1) and H had a different mother (m2). The question to be resolved was whether the father (f) of H was also the father of R and/or K.
This was a deficiency case, because only the 3 women were available for investigation. The case could only be solved by ChrX marker testing. In addition to the markers from the Investigator Argus X12 kit, we used 11 other ChrX markers, and considered haplogroups in the interpretation of the results. We found one complete X-chromosomal concordance over all of the tested markers between R and K, and one between K and H, but there was no X-chromosomal concordance between R and H.
X-chromosomal conformity in women is explainable in two ways. They either have an identical paternal X-chromosome, because a man only has one X-chromosome to pass down to all his daughters, or the same maternal X-chromosome. Therefore, we could deduce that the X-chromosome agreement between H and K must have resulted from them having the same father. Because there was no X-chromosome conformity between R and H, these women must have had different fathers. The X-chromosomal concordance between R and K results from their having the same maternal X-chromosome.
What is the next development in ChrX marker application in kinship testing?
We are currently investigating ChrX-Indel polymorphisms, which are underutilized in forensic science. Indel polymorphisms can solve kinship tests in the case of stains or skeletal remains more successfully, because primers can be designed to amplify very short fragments.
Dr. Edelmann, thank you very much for your time.
