Application of massively parallel sequencing (MPS) in paternity testing - case report.

Aim of the study: We present the application of massively parallel sequencing (MPS) to extend the scope of analysis in a disputed paternity case. Material and methods: A standard paternity test comprising 16 autosomal STRs was performed by capillary electrophoresis (CE) using 3130xl Genetic Analyzer. Additionally, MPS was performed with ForenSeq DNA Signature Prep Kit and Illumina MiSeq FGx™ Forensic Genomics System. Paternity index (PI) was calculated using DNAStat v.2.1 software. Results>: CE revealed two mismatches, at D21S11 and VWA, between the putative father and the child. Based on MPS results, the mismatches were analyzed and a nonconsensus sequence of allele 14 at the VWA locus in the mother - child pair was identified. Different sequence variants were also detected in 16-16 homozygote alleles at the D3S1358 locus in the child. Conclusions: MPS helped to formulate a definite conclusion regarding the paternity of the defendant and provided full information on intra-allelic polymorphism.

The influence of renal function on the association of rs854560 polymorphism of paraoxonase 1 gene with long-term prognosis in patients after myocardial infarction.

Paraoxonase 1 (PON1) is an enzyme responsible for the antioxidant properties of high density lipoprotein (HDL). The activity of PON1 is decreased in patients with coronary artery disease, myocardial infarction or chronic kidney disease. rs662 and rs854560 are single nucleotide polymorphisms (SNPs) associated with PON1 activity and 10-year cardiovascular mortality of patients with stable coronary artery disease. We investigated the association of rs662 and rs854560 SNPs of the PON1 gene with 5-year mortality in patients with ST-elevation myocardial infarction (STEMI) treated invasively. We analyzed the data of consecutive patients with STEMI treated with primary PCI. Genotyping was performed with the TaqMan method. The analyzed end-point was total 5-year mortality. Additional subgroup analysis was performed for survival of patients depending on their eGFR. The study group comprised 634 patients (mean age 62.3 ± 11.85 years; 25.2% of women, n = 160; PCI successful in 92.3%, n = 585). No clinically relevant differences in baseline characteristics were found between the genotypes. No association between either genotype and 5-year mortality was found: p = 0.4 for the rs662 SNP, p = 0.73 for the rs854560 one (log-rank test). However, in a subgroup of patients with eGFR below median value (78.6 ml/min/1.73m2) the rs854560 AA homozygotes had a significantly lower probability of survival (p = 0.047, log-rank test). The AA genotype of the rs854560 SNPs of the PON1 gene is associated with increased mortality in patients after myocardial infarction in the subpopulation of patients with lowered eGFR. This phenomenon may be explained by potentially lower PON1 activity in kidney disease.

The 9p21 polymorphism is linked with atrial fibrillation during acute phase of ST-segment elevation myocardial infarction.

The aim of the study was to find whether patients carrying polymorphic allele of the rs10757278 polymorphism from 9p21 locus have changed risk of arrhythmia (atrial fibrillation, AF; sustained ventricular tachycardia or ventricular fibrillation, sVT/VF) during acute phase of myocardial infarction. Retrospective analysis of data collected prospectively from two independent centers was performed. The clinical data were pooled from two independent cardiac registries: (1) the Warsaw ACS genetic registry (STEMI and NSTEMI/UA patients hospitalized in the years 2008-2011; only STEMI patients were analyzed); (2) the Bialystok STEMI genetic registry (STEMI patients hospitalized in years 2001-2005, who survived the first 48 h from hospital admission). Data regarding sVT/VF and AF within first 24 h were analyzed. The patients were genotyped with rs10757278 polymorphism. 1083 patients were included in the analysis; 62 (5.7 %) patients had sVT/VF during acute phase and 78 (7.2 %) patients had AF, 46 (4.2 %) patients had new-onset AF. Minor allele frequency in all patients with AF was significantly different from those without AF (0.40 vs 0.51, p = 0.0096). When only new-onset AF was analyzed, the trend was the same, with significant protective effect in recessive model [OR 0.41 (95 % CI 0.17-0.97), p = 0.025]. The effect was independent of age and GRACE score. No relationship was found between sVT/VF and rs10757278. Patients with STEMI, who survived until hospitalization with polymorphic allele of 9p21 rs10757278 SNP have less AF during acute phase of STEMI. SNP rs10757278 is not linked with sVT/VF in acute phase of STEMI.

Changes in Surface Charge Density of Blood Cells in Fatal Accidental Hypothermia.

The objective of this research was to evaluate postmortem changes concerning electric charge of human erythrocytes and thrombocytes in fatal accidental hypothermia. The surface charge density values were determined on the basis of the electrophoretic mobility measurements of the cells conducted at various pH values of electrolyte solution. The surface charge of erythrocyte membranes after fatal accidental hypothermia increased compared to the control group within whole range of experimental pH values. Moreover, a slight shift of the isoelectric point of erythrocyte membranes towards high pH values was observed. The surface charge of thrombocyte membranes in fatal accidental hypothermia decreased at low pH compared to the control group. However, at pH range 4-9, the values increased compared to the control group. The isoelectric point of thrombocyte membranes after fatal accidental hypothermia was slightly shifted towards low pH values compared to the control group. The observed changes are probably connected with the partial destruction and functional changes of the blood cell structure.

Population genetics of 30 INDELs in populations of Poland and Taiwan.

The Investigator DIPplex(®) kit (Qiagen) contain components for the simultaneous amplification and analysis of 30 biallelic autosomal INDELs and amelogenin. The objective of this study was to estimate the diversity of the 30 markers in Polish (N P = 122) and Taiwanese (N T = 126) population samples and to evaluate their usefulness in forensic genetics. All amplicon lengths were shorter than 160 base pairs. The DIPplex genotype distributions showed no significant deviation from Hardy-Weinberg rule expectations (Bonferroni corrected) except for DLH39 in the Taiwanese population. Among the Poles and the Taiwanese the mean observed heterozygosity values are 0.4385 and 0.4079, and the combined matching probability values are 7.98 × 10(-14) and 1.22 × 10(-11), respectively. The investigated marker set has been confirmed as a potential extension to standard short tandem repeat-based kits or a separate informative system for individual identification and kinship analysis. Eight INDELs have been selected as possible ancestry informative single-nucleotide polymorphisms for further analyses.

Y-STR data of the Yfiler Plus panel in population of north-eastern Poland. / Dane populacyjne dla markerów zestawu Yfiler Plus w populacji Polski pólnocno-wschodniej.

Background: The use of new high-resolution and forensic identification capabilities for population studies offered by new multiplex methods (such as Yfiler Plus) is crucial in forensic genetics cases. The development of haplotype frequency databases is essential to take full advantage of the new Y chromosome determination capabilities. Purpose: Development of the haplotype database of the Yfiler Plus kit for a population-based sample of 534 males from northeastern Poland and calculation of suitability parameters for forensic genetics studies. Materials and methods: The study was conducted on a population sample of 534 unrelated males from the area of northeastern Poland using the Yfiler Plus panel of 27 markers located on the Y chromosome. Results: Four haplotypes appeared twice. The Discrimination Capacity (DC) of the entire set was 0.9925. The highest Gene Diversity (GD) value was calculated for DYS518 (0.86) belonging to the fast-mutation markers, while the lowest GD was calculated for DYS392 (0.42). Conclusion: The results indicate the need for further research and observation of changes, both in different regions of Poland and across Europe.

Population Genetic Data of 30 Insertion-Deletion Markers in the Polish Population.

(1) Background: Insertion-deletion (InDel) markers show the advantages of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) and are considered alternative markers in forensic genetics. (2) Methods: Allelic frequencies and corresponding forensic efficiency parameters of 30 autosomal polymorphic InDel loci included in the Investigator DIPplex kit (Qiagen) were obtained in a sample of 631 unrelated Polish individuals. Allelic frequency data were compared with those reported for selected populations (3) Results: All the loci conformed with Hardy-Weinberg equilibrium after applying a Bonferroni correction and no pair-wise significant linkage disequilibrium was detected. (4) Conclusions: DIPplex Kit differences were high among populations worldwide. The InDel markers are highly discriminating for human identification purposes in the Polish population.

[Validation and evaluation of a five miniSTRs kit in forensic genetics]. / Walidacja i ocena przydatnosci zestawu pieciu markerów miniSTR w genetyce sadowej.

The newly designed and optimized miniplex contains the following markers: D3S3053, D6S474, D9S2157, D20S482 and sex-determining marker - amelogenin. The target amplicon lengths for the developed multiplex are 71-135 bp. Amplification products were detected in a fluorescence based automated genetic analyzer. A minimal DNA sample required to obtain full genetic profiles was 250 pg. The usefulness of these miniSTRs in genotyping of severely degraded forensic samples, such as stains of blood and semen, saliva on cigarette butts and telogen hair has been confirmed in validation studies. The designed pentaplex offers a new potential screening tool in cases of old crime scenes, mass disasters, mass graves, etc., where DNA degradation, body fragmentation or large numbers of victims occur. The use of additional non-CODIS markers may increase typeability of severely degraded samples and ensure a higher potential for genetic discrimination.

[Evaluation of visualization of biological stains with the use of alternative light source (ALS) for the purpose of genetic identification. Part II. Semen samples analysis]. / Ocena wizualizacji sladów biologicznych z uzyciem alternatywnego zródla swiatla (ALS) w aspekcie identyfikacji genetycznej. Czesc II. Analiza sladów nasienia.

Detection of seminal stains on items such as clothing and bedding is a significant element of investigation in sexual assault cases. The use of alternative light source may assist in their identification. The objective of the investigation was the evaluation of human semen visualization with the use of alternative light source for the purpose of genetic identification. The tests demonstrated that experimentally prepared semen stains on the bright base could be best seen in the natural light and white light when the semen was diluted at a ratio 1:10. The complete typeability of AmpFISTR SGM Plus kit loci was evaluated in semen which was diluted at a ratio 1:1750 and typeability of AmpFISTR SGM Plus kit loci was incomplete in semen diluted at a ratio 1:2000. After washing with laundry detergents, semen stains were still recognizable under ALS wavelength 455 nm, while wearing orange goggles.

[Evaluation of visualization of biological stains with the use of alternative light source (ALS) for the purpose of genetic identification. Part I. Blood and saliva stains analysis]. / Ocena wizualizacji sladów biologicznych z uzyciem alternatywnego zródla swiatla (ALS) w aspekcie ich identyfikacji genetycznej. Czesc I. Analiza sladów krwi i sliny.

The objective of the investigation was evaluation of visualization of human blood and saliva stains with the use of alternative light source for the purpose of genetic identification. Experimental bloodstains on the bright base were the most clearly seen in the natural light and white light, up to blood dilution of 1:600. Complete typeability of AmpFISTR SGM Plus kit profiles was obtained from bloodstains at dilution 1:1500. Partial AmpFISTR SGM Plus kit profiles were typed from bloodstains at dilutions 1:1750 and 1:2000. Experimental saliva stains on the light-colored base were completely invisible in the natural light and white light, while they were visualized at wavelength range 300-415 nm through yellow goggles, and at wavelength range 300-455 nm through orange goggles at saliva dilution 1: 600. Complete typeability of AmpFISTR SGM Plus kit loci was obtained from saliva stains at dilution 1:1750. Partial AmpFISTR SGM Plus kit profiles were typed from saliva stains at dilution 1:2000. The wavelength of 455 nm and orange goggles were the optimal set for visualization of bloodstains on various, noncontrasting materials. Other useful wavelength/combinations of goggles were CSS light/red goggles. In case of saliva, the most useful general condition for visualization of stains on various, non-contrasting materials was with the wavelength set to 300-415 nm, while wearing yellow goggles. Other useful combinations of wavelength/goggles were 300-455 nm/orange or red goggles, and also CSS light/orange or red goggles.

[Polymorphism of 11 non-CODIS STRs in a population sample of ethnic minority of Polish Tatars residing in northeastern Poland]. / Polimorfizm 11 loci STR nie objetych systemem CODIS w próbce populacyjnej Tatarów Polskich zamieszkujacych teren Podlasia.

Population genetic data for 11 STRs included in the Humantype Chimera kit were obtained by multiplex PCR and subsequent automated fluorescent detection (ABI 310) from a sample of 125 unrelated individuals of ethnic minority of Polish Tatars residing in Podlasie Region (NE Poland). The genotype distributions conformed to HWE for all the analyzed loci except D2S1360 and D21S2055. The highly polymorphic systems exhibit high informativeness and are a potential extension to CODIS loci.

Genetic identification of a gunshot victim four years posthumously.

The paper presents a personal identification case of an unrecognized corpse, presumably belonging to a male missing for four years. The cadaver was buried in a ground ditch and covered with slaked lime and soil. During the investigation the burial place was indicated. The corpse was exhumed and afterwards transferred to the Department of Forensic Medicine, Medical University of Bialystok. External examination and autopsy findings demonstrated adipocere formation and putrefaction, as well as two gunshot wounds in the thorax and the head assumed to be the cause of death. Personal identification procedure included skeletal and dental examination. As a source material for genetic typing, the femur, brain, lung, kidney and spleen samples were collected. DNA templates were extracted by a modified organic procedure and genotyped with the use of AmpFISTR Identifiler Amplification Kit and PowerPlex Y System in an ABI 310 Genetic Analyzer (Applied Biosystems). All the soft tissue samples yielded sufficient quantity and quality of DNA to perform genetic profiling.

Detectability of SGM Plus profiles in heart and lungs tissue samples incubated in different environments.

The aim of the study was assessment of environmental effect on typeability of AmpFlSTR SGM Plus loci: D3S1358, VWA, D16S539, D2S1338, D81179, D21S11, D18S51, D19S433, TH01, FGA and gender marker amelogenin. Heart and lungs specimens collected during autopsies of five persons aged 20-30 years were incubated at 21 degrees C and 4 degrees C in different environmental conditions, fresh different water and soil conditions. DNA was extracted by organic method from tissue samples collected in 7-day intervals and subsequently typed using AmpFlSTR SGM Plus kit and ABI 310. Incubation at 21 degrees C and prevented air access, as well as in peat soil and in sand favoured faster DNA degradation reflected by decrease in typeability rate. In samples with negative genotyping results no DNA was found by fluorometric quantitation. Decomposed soft tissues are potential material for DNA typing.

[Ethnic variation and forensic usefulness of Y-STR loci in inhabitants of northeastern Poland]. / Polimorfizm loci Y-STR wsród ludnosci Polski pólnocno-wschodniej w aspektach zróznicowania etnicznego i przydatnosci w badaniach medyczno-sadowych.

The objective of the investigation was the calculation of biostatistical indices and parameters of medicolegal usefulness and extension of the knowledge on the genetic structure of the population in view of its historical background and ethnic composition. Polymorphism of Y-STR loci was determined in population samples including the total of 718 males of Polish nationality and belonging to the minorities of Byelorussians, Lithuanians, Polish Tatars and the religious minority of the Old Believers. Statistical analysis of genetic polymorphisms indicated their usefulness in characterizing populations and ethnic groups. The variations in haplotype distribution in northeastern Polish populations should be taken into consideration while evaluating probability of genetic profile matching in medicolegal expert opinions.

[Polymorphism of 11 autosomal STRS in a population sample of Belarussian minority residing in the region of Podlasie (Northeastern Poland)--an extension of the STR core set]. / Polimorfizm 11 autosomalnych loci STR w populacji bialoruskiej mniejszosci narodowej zamieszkujacej region Podlasia--rozszerzenie typowego panelu badawczego.

Population genetic data for 11 STRs included in the Humantype Chimera kit were obtained by multiplex PCR and subsequent automated fluorescent detection (ABI 310) from a sample of 200 unrelated individuals of both genders belonging to the Belarussian minority residing in the region of Podlasie (Northeastern Poland). The objective of the investigations was determination of 11 STRs frequency and calculation of parameters of their usefulness in medicolegal examinations. The genotype distributions conformed to HWE for all the analyzed loci. The highly polymorphic systems exhibit a high degree of informativeness and are a potential extension to CODIS loci, particularly in kinship analysis and deficiency cases.

Analysis of paternity exclusions in the material collected by the Department of Forensic Medicine, Medical University of Bialystok in 2008-2017.

AIM OF THE STUDY: Analysis of frequency and structure of paternity exclusions in the material collected by the Department of Forensic Medicine, Medical University of Bialystok in 2008-2017. MATERIAL AND METHODS: The paper is based on paternity test reports involving alleged father-child-mother trios. In a total of reviewed 958 cases, 187 exclusions were identified. The analysis was carried out on the basis of the results of DNA tests. DNA extraction was performed using QIAamp DNA Mini Kit (Qiagen) and DNA quantitation using Quantifiler Human DNA Quantification Kit and 7500 Real-Time PCR System (Applied Biosystems). AmpFLSTR Identifiler PCR Amplification Kit and a PCR System 9700 thermal cycler (Applied Biosystems) were used for DNA amplification. RESULTS: Over the analyzed period, the number of paternity tests was nearly halved, whereas the percentage of exclusions in individual years varied significantly (33.9-13.3%), with the average of 26.3%. The highest efficiency of exclusions was observed for D18S51 (0.7166) and FGA (0.7059), and the least effective system was TPOX (0.3048). CONCLUSIONS: The applied set of markers has been demonstrated to be an efficient tool in genetic paternity tests in the context of the recommended rules of exclusion.

Contrast-enhanced ultrasonography in diagnosing liver metastases.

BACKGROUND: Dual-phase spiral computed tomography (CT) is still the primary imaging technique in the diagnosis of focal liver lesions. Contrast-enhanced ultrasonography (CEUS) is the most sensitive sonographic technique. The purpose of this study was to investigate the efficacy of CEUS in detecting liver metastases compared with CT as the standard of reference. MATERIAL/METHODS: The examined group consisted of 51 patients (24 men and 27 women, age range: 27-84 years, mean: 57.4 years) suspected of liver metastases. The routine diagnostic approach consisted of B-mode US, CEUS, and CT. Final diagnosis was made at cytologic (n=18) or histologic examination (n=14) and in 9 patients by combining information from CT scans, medical history, and clinical and biochemical investigations. RESULTS: Liver cysts and abscesses were detected in 10 patients. They were excluded from the further analyses. In the remaining 41 patients a total of 134 metastases were detected. In 15 patients with metastases, US images of the liver appeared normal. CEUS detected metastases in 36 patients. The sensitivities of the methods per patient were US 63.4% and CEUS 90.2%. Sensitivities of the methods per lesion were US 60.9%, CT 77.6%, and CEUS 90.2%. Application of contrast media (SonoVue) significantly increased diagnosing of liver metastases compared with standard sonography and CT. CONCLUSIONS: CEUS increased diagnostic confidence in the detection and characterization of hepatic metastases compared with standard sonography. Real-time contrast-enhanced sonography is particularly advantageous in detecting small metastases.

Vitreous humour as a potential DNA source for postmortem human identification.

PURPOSE: The aim of this study was the assessment of vitreous humor as a potential DNA for forensic human postmortem identification. MATERIAL AND METHODS: Vitreous humor samples were collected using two alternative approaches from 25 corpses of either sex during autopsies. DNA was extracted by standard organic method. Recovered DNA was quantitiated fluorometrically. AmpFlSTR SGM Plus kit and ABI 310 Genetic Analyzer (Applera) were used to obtain genetic profiles. RESULTS: Different DNA yields were quantitated in vitreous body depending on cause of death and sampling approach. CONCLUSION: Vitreous humor is a potential DNA for forensic human postmortem identification depending on a sampling method used.

Typeability of AmpFlSTR SGM Plus loci in brain and thyroid gland tissue samples incubated in different environments.

Autolysis and putrefaction are crucial factors responsible for degradation of cells, tissues, and organs. Postmortem changes may assume different course depending on extrinsic and intrinsic conditions. The aim of the study was assessment of environmental effect on typeability of AmpFlSTR SGM Plus loci: D3S1358, VWA, D16S539, D2S1338, D81179, D21S11, D18S51, D19S433, TH01, FGA, and gender marker amelogenin. Brain and thyroid gland tissue specimens collected during autopsies of five persons aged 20-30 years were incubated at 21 degrees C and 4 degrees C in different environmental conditions. DNA was extracted by organic method from tissue samples collected in 7-day intervals and subsequently typed using AmpFlSTR SGM Plus kit and ABI 310. A fast decrease in typeability rate was seen in specimens incubated in peat soil and in sand. Brain tissue samples were typeable in all AmpFlSTR SGM Plus loci within 126 days of incubation at 4 degrees C. Faster DNA degradation was recorded in thyroid gland specimens. In samples with negative genotyping results, no DNA was found by fluorometric quantitiation.

[Polymorphism of 10 autosomal Strs in a population sample of Podlasie (NE Poland)--an extension to the STR core set]. / Polimorfizm 10 autosomalnych loci STR w populacji podlasia--rozszerzenie typowego panelu badawczego.

Population genetic data for 10 STRs included in the Humantype Chimera kit were obtained by multiplex PCR and subsequent automated fluorescent detection (ABI 310) from a sample of 220 unrelated individuals of Polish ancestry residing in north-eastern Poland. The genotype distributions conformed to HWE for all the analysed loci. The highly polymorphic systems exhibit a high informativeness and may be helpful in cases requiring an extension of the CODIS loci system, particularly in kinship analysis and deficiency cases.