Andrzej Ossowski

Example of human individual identification from World War II gravesite

This paper presents the procedure elaborated by our team which was applied to the mode of identification of Red Army soldiers who were taken as prisoners by the German Army during World War II and deceased in captivity. In the course of our search the unmarked burial of ten Soviet prisoners of war was found. Historical, anthropological and genetic research conducted by us led to the personal identification of nine of them, including two by means of DNA analysis.

CAG Repeat Polymorphism in the Androgen Receptor Gene in Women with Rheumatoid Arthritis

Objective. Rheumatoid arthritis (RA) is the most common chronic, autoimmune, inflammatory disease, with a genetic and hormonal background. The prevalence of women among patients with RA suggests the important role of sex hormones in the pathogenesis of RA. We examined the association between CAG repeat polymorphism in the androgen receptor (AR) gene and susceptibility to RA and its clinical features in white women. Methods. The study groups consisted of 325 female patients with RA and 238 female controls. CAG repeat polymorphism was determined using polymerase chain reaction and subsequent fragment analysis by capillary electrophoresis. Results. The number of CAG repeats in patients did not differ from that of controls (22.1 ± 2.9 vs 21.9 ± 2.9, respectively; p = 0.26), but the presence of articular erosions was associated with a lower number of repeats in the shorter allele of patients with RA (20.4 ± 2.2 vs 21.2 ± 2.4; p = 0.031). When alleles with < 22 CAG were classified as short (S) and those with ≥ 22 CAG as long (L), the age at diagnosis of RA was lower in women with S-S genotype in comparison to combined S-L + L-L genotypes (43.0 ± 14.6 yrs vs 47.6 ± 12.5 yrs; p = 0.021). In patients with the L-L genotype, the frequency of erosive disease (OR 0.45, 95% CI 0.25–0.80, p = 0.0085) and extraarticular manifestations (OR 0.50, 95% CI 0.26–0.98, p = 0.047) was lower in comparison to carriers of the S allele. In multivariate analysis, the L-L genotype was an independent factor associated with a lower risk of erosions (OR 0.44, 95% CI 0.22–0.90, p = 0.024). Conclusion. The results suggest the association of short AR (CAG)n alleles with earlier onset and a more aggressive course of RA.

The Polish Genetic Database of Victims of Totalitarianisms

This paper describes the creation of the Polish Genetic Database of Victims of Totalitarianism and the first research conducted under this project. On September 28th 2012, the Pomeranian Medical University in Szczecin and the Institute of National Remembrance-Commission for Prosecution of Crimes against the Polish Nation agreed to support the creation of the Polish Genetic Database of Victims of Totalitarianism (PBGOT, www.pbgot.pl). The purpose was to employ state-of-the-art methods of forensic genetics to identify the remains of unidentified victims of Communist and Nazi totalitarian regimes. The database was designed to serve as a central repository of genetic information of the victims DNA and that of the victims nearest living relatives, with the goal of making a positive identification of the victim. Along the way, PGBOT encountered several challenges. First, extracting useable DNA samples from the remains of individuals who had been buried for over half a century required forensic geneticists to create special procedures and protocols. Second, obtaining genetic reference material and historical information from the victims closest relatives was both problematic and urgent. The victims nearest living relatives were part of a dying generation, and the opportunity to obtain the best genetic and historical information about the victims would soon die with them. For this undertaking, PGBOT assembled a team of historians, archaeologists, forensic anthropologists, and forensic geneticists from several European research institutions. The field work was divided into five broad categories: (1) exhumation of victim remains and storing their biological material for later genetic testing; (2) researching archives and historical data for a more complete profile of those killed or missing and the families that lost them; (3) locating the victims nearest relatives to obtain genetic reference samples (swabs), (4) entering the genetic data from both victims and family members into a common database; (5) making a conclusive, final identification of the victim. PGBOTs first project was to identify victims of the Communist regime buried in hidden mass graves in the Powązki Military Cemetery in Warsaw. Throughout 2012 and 2013, PGBOT carried out archaeological exhumations in the Powązki Military Cemetery that resulted in the recovery of the skeletal remains of 194 victims in several mass graves. Of the 194 sets of remains, more than 50 victims have been successfully matched and identified through genetic evidence.

Comparison of three different DNA extraction methods from a highly degraded biological material

The identification of unknown victims is one of the most challenging tasks faced by forensic medicine. This is due to the rapid decomposition of tissues, beginning at the moment of death and caused by released enzymes and microbial activity. Decay is directly associated with the decomposition of soft tissues and also the degradation of genetic material inside cells. Decomposition rates vary depending on a number of environmental factors, including temperature, humidity, season, and soil properties. Decomposition also differs between bodies left in the open air or buried. To date, forensic medicine has identified mainly people who were the victims of various types of criminal offences. However, with advances in identification methods, increasingly frequent attempts are made to identify the victims of armed conflicts, crimes of totalitarian regimes, or genocide. The aim of the study was to compare three different methods for the extraction of nuclear DNA from material considered in forensic medicine as difficult to handle, i.e. fragments of bones and teeth, and to determine the performance of these methods and their suitability for identification procedures.

Population genetics of 15 autosomal STR loci in the population of Pomorze Zachodnie (NW Poland)

Allele frequency data and forensic efficiency parameters for 15 STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA were estimated from a sample of 600 unrelated individuals from the Pomorze Zachodnie (NW Poland). The combined MP and PE for all 15 loci are 3.9x10(-18) and 0.9999988, respectively. Pairwise comparisons between Northwestern Poland and other Polish populations were performed.

A case study of an unknown mass grave — Hostages killed 70 years ago by a Nazi firing squad identified thanks to genetics

Almost 6 million people died in Poland during the Nazi occupation and about 570 thousand during the Soviet occupation. But the end of the war was not the end of the trauma. Historians estimate that at least 30 thousand people were killed during the Stalinist regime in Poland. In 2012 the Institute of National Remembrance started to search for hidden burials of victims of communism. Many exhumations were carried out under the project. One of them took place in Białystok, eastern Poland. According to information gathered by local historians, a detention centre in the heart of city was the place of secret burials of victims of the communist regime. During the exhumation work a burial pit with the remains of 24 victims was found. Its characteristics supported the hypothesis that these people were shot on the spot, in a mass execution during the Nazi occupation. Historians knew of only one such execution, but its victims - according to the available records - were supposed to have been exhumed at the end of the war. Exhumation works and the discovery of the discussed mass grave put in question the events of 1944, which would have been impossible without the field work. The first identifications confirmed the doubts of historians, since both the results of genetic profiling and the conducted anthropological analysis revealed that at the end of the war a mistake was made, and bodies other than those suspected had been exhumed. Having established this fact, the mass grave created at that time should be investigated to reveal the identity of the remains uncovered then.

Y-chromosomal haplotypes for the AmpFlSTR Yfiler PCR amplification kit in a population sample of Bedouins residing in the area of the Fourth Nile Cataract

The Bedouin are a part of a predominantly desert-dwelling Arab ethnic group traditionally divided into tribes or clans. The term ‘Bedouin’ means, ‘those who live in bādiyah’ or ‘those who live in the desert’. Altogether, the Bedouin population in Africa and in the Middle East numbers about 4 million. The contemporary Bedouin tribes have originated from the area of Arabian Peninsula and overlapped older Nubian populations of Christian Era that had developed after Marawy culture which was an extension to the Pharaonic culture. Anthropological studies on the native inhabitants dwelling in region of the 4th Nile Cataract revealed significant morphologic variation based on cephalometric and somatoscopic data. The skin color ranges from very dark, similar to that seen in inhabitants of Central Africa, to olive – typical for the Mediterranean race (prof. Jan Czekanowski – personal communication). Examples of facial types among individuals dwelling in the same village are presented in Supplementary Fig. 1. This dissimilarity results obviously from history of the tribes involved in banditry and trade of slaves from southern Sudan and other parts of Africa, since many of the slaves assimilated into the local population. Additionally, islamisation and arabisation of Sudan caused admixture to the pool of resident Christian inhabitants originating from the ancient Meroe and Nubia. Currently detailed genetic investigations are performed on these populations as well as on archeological sites (burial grounds) to learn more about the origin of the inhabitants. A population sample from four indigenous Bedouin groups: Shaghiya, Manasir, Ababda and Hassaniya, was examined for Ychromosome haplotype diversity. The areas inhabited by the tribes are marked on the map of the area of the Fourth Nile Cataract in the Manasir Desert (Supplementary Fig. 2). To our knowledge, genetic data on Bedouin inhabitants of Sudan has not been included in the literature so far. DNA from 64 unrelated Bedouin males was extracted from mouth swab samples using the QIAamp DNA Mini Kit (Qiagen). DNA was quantitated spectrophotometrically. 1–2 ng target DNA was amplified according to the AmpFlSTR Yfiler PCR Amplification Kit User Manual, Rev B (Applied Biosystems, USA) in PCR System 9700 (Applied Biosystems, USA) using AmpFlSTR Yfiler PCR Amplification Kit [1]. Electrophoresis and typing were performed in the ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, USA) using denaturing polymer POP-4. GeneScan 500 LIZ was used as the internal lane standard. Reference sequenced ladder included in the kit was used for genotype classification. The 17 Y-STR results were analyzed using GeneMapper ID v.3.2 (Applied Biosystems, USA).

Population analysis and forensic evaluation of 21 autosomal loci included in GlobalFiler™ PCR Kit in Poland

The allele distribution and biostatistical parameters of a Polish population from the central part of Europe were evaluated by forensic application of a GlobalFiler TM PCR kit. DNA was obtained from 600 unrelated individuals with Polish parents from all over Poland. The DNA was deposited in the Polish Genetic Database of Totalitarianism Victims (PBGOT). The investigated population sample met the criteria for independent inheritance. The distributions of the genotype frequencies for all of the investigated 21 autosomal short tandem repeat (STR) loci in the Polish population were in Hardy-Weinberg’s equilibrium. The observed heterozygosity (HET) ranged from 0.617 to 0.962, power of discrimination (PD) was from 0.785 to 0.994, and power of exclusion (PE) was from 0.311 to 0.922, leading to accumulated values of PD=0.99999999999999999999999998 and PE=0.99999999899. The overall matching probability (MP) value was extremely low, i.e., 1.8x10. The GlobalFiler PCR Amplification Kit is a valuable tool for genetic profiling in forensics in Poland.

Genetic Identification of Communist Crimes' Victims (1944-1956) Based on the Analysis of One of Many Mass Graves Discovered on the Powazki Military Cemetery in Warsaw, Poland.

As the result of the communist terror in Poland, during years 1944–1956 more than 50,000 people died. Their bodies were buried secretly, and most places are still unknown. The research presents the results of identification of people buried in one of many mass graves, which were found at the cemetery Powązki Military in Warsaw, Poland. Exhumation revealed the remains of eight people, among which seven were identified genetically. Well‐preserved molars were used for the study. Reference material was collected from the closest living relatives. In one case, an exhumation of victims parents had to be performed. DNA from swabs was extracted with a PrepFiler® BTA Forensic DNA Extraction Kit and organic method. Autosomal, Y‐STR amplification, and mtDNA sequencing were performed. The biostatistical calculations resulted in LR values from 1608 to 928 × 1018. So far, remains of more than 50 victims were identified.

Role of platelets in the modulation of kidney allograft recipients’ immune systems

BACKGROUND Renal transplantation is the most effective method of treatment in end-stage renal disease. Chronic allograft rejection still remains a challenge for transplant physicians. Despite a growing amount of data regarding the role of platelets (PLT) in immunological processes, few reports have correlated number of platelets with transplanted kidney function. We aimed to evaluate the correlation between number of circulating platelets and number of immune system cells, including lymphocytes CD 4+, CD8+, lymphocytes B, monocytes, NK cells, and lymphocytes T reg in kidney transplant recipients with stable graft function. MATERIAL AND METHODS We enrolled 100 kidney transplant recipients (ages 20-78 years) 10 month to 10 years after transplantation. The numbers of platelets (using standard procedure) and immune blood cells were evaluated using flow cytometry. Statistical analysis was performed with Spearman rank correlation. RESULTS We found a negative correlation between number of platelets and number of lymphocytes T reg, and a positive correlation between platelet count and number of other examined immunocompetent cells. CONCLUSIONS The number of PLT correlates with number of cells responsible for induction and effector mechanisms of acquired cellular response.