Canine STR analyses in forensic practice. Observation of a possible mutation in a dog hair.

In a case of the death of a 7-year-old boy, the police investigations revealed a possible dog attack contrary to the witness testimonies. DNA investigations were carried out from hairs, saliva and bloodstains with 10 canine-specific STR loci by the use of fluorescently labelled multiplex PCR and the ABI PRISM 310 genetic analyzer. The analysis of one hair sample revealed one allele deviation from the profile of the putative Rottweiler perpetrator possibly caused by a mutation. The PCR fragments in question at the PEZ20 locus were sequenced and compared with the alleles detected in the Hungarian canine population and identified on a repeat number basis. The allele frequencies were determined based on typing of 242 genetically independent canine individuals from 72 breeds. The results suggested that two of the canine individuals could be the perpetrators.

Canine microsatellite polymorphisms as the resolution of an illegal animal death case in a Hungarian zoological gardens.

Several animal carcasses were found in the paddocks of a Hungarian County Zoo during 1 week. The 14 animals killed were thought to be the victims of a dogfight training. The primary suspect was the security guard of the Zoo with his guard dogs. DNA tests were carried out on hairs and bloodstains and 10 canine-specific STR loci were analysed by fluorescently labelled multiplex PCR using the ABI PRISM 310 Genetic Analyzer. The results confirmed that the killer was a single animal and all of the guard dogs were excluded.

Variations in primer sequences are the origin of allele drop-out at loci D13S317 and CD4.

STRs have become almost the exclusive tool of genetic scientists in forensic typing work. Consequently, large numbers of samples are genotyped and the detection of rare abnormalities is to be expected. We found rare losses of alleles, also known as drop-out, at the two STR loci D13S317 and CD4. Drop-out at D13S317 was accidentally found in typing of suspects in a murder case and three other examples of drop-out were found at locus CD4 during paternity testing. The lost alleles reappeared when alternative PCR primer pairs were used. Sequences of lost alleles were characterised at the molecular level after cloning. Variations were found in the primer sequences and these are believed to prevent amplification or to reduce amplification yield and to be the origin of the allele drop-out.

Online reference database of European Y-chromosomal short tandem repeat (STR) haplotypes.

The reference database of highly informative Y-chromosomal short tandem repeat (STR) haplotypes (YHRD), available online at http://ystr.charite.de, represents the largest collection of male-specific genetic profiles currently available for European populations. By September 2000, YHRD contained 4688 9-locus (so-called "minimal") haplotypes, 40% of which have been extended further to include two additional loci. Establishment of YHRD has been facilitated by the joint efforts of 31 forensic and anthropological institutions. All contributing laboratories have agreed to standardize their Y-STR haplotyping protocols and to participate in a quality assurance exercise prior to the inclusion of any data. In view of its collaborative character, and in order to put YHRD to its intended use, viz. the support of forensic caseworkers in their routine decision-making process, the database has been made publicly available via the Internet in February 2000. Online searches for complete or partial Y-STR haplotypes from evidentiary or non-probative material can be performed on a non-commercial basis, and yield observed haplotype counts as well as extrapolated population frequency estimates. In addition, the YHRD website provides information about the quality control test, genotyping protocols, haplotype formats and informativity, population genetic analysis, literature references, and a list of contact addresses of the contributing laboratories.

Analysis of eight STR loci in two Hungarian populations.

A multiplex reaction for the eight STR loci D3S1358, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 was used to generate allele frequency databases for two Hungarian population samples, Caucasians from the Budapest area and Romanies from Baranya county. During the analysis two intermediate-sized alleles and a sequence variant allele were observed at the D7S820 locus. All three types of allelic variants were found to have modifications in the same block of a (T)9 stretch located within the 3' flanking region of each allele, which may indicate a possible higher mutation rate of this (T)9 block. For the loci D3S 1358 and D7S820 the Romany population database showed departures from Hardy-Weinberg equilibrium. The forensic efficiency values for the Romany population were slightly different from those found in the Hungarian Caucasian population. Comparing the allele frequency values by G-statistic, calculating the F(ST) indices and with the pair-wise comparisons of interpopulation variance, the two Hungarian populations could be distinguished using data from the eight STR loci.

Analysis of eight STR loci in two Hungarian populations.

A collection of eight STR loci (D3S1358, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) was used to generate allele frequency databases for two Hungarian population samples, Caucasians from the Budapest area and Romanies from Baranya county. During the analysis two intermediate sized alleles and a sequence variant allele were observed at the D7S820 locus. All three types of allelic variants were found to have modification (deletion, insertion, transversion) in the same block of a (T)(9) stretch located within the 3' flanking region of each allele, which may indicate a possible higher mutation rate of this (T)(9) block. For the loci D3S1358 and D7S820 the Romany population database showed departures from Hardy-Weinberg equilibrium. The forensic efficiency values for the Romany population were slightly different from those found in the Hungarian Caucasian population. Comparing the allele frequency values by G-statistic, calculating the F(st) indices and with the pairwise comparisons of inter-population variance, the two Hungarian populations could be distinguished using data of the eight STR loci.

A new method for the evaluation of matches in non-recombining genomes: application to Y-chromosomal short tandem repeat (STR) haplotypes in European males.

A 9-locus microsatellite framework (minimal haplotype), previously developed for forensic purposes so as to facilitate stain analysis, personal identification and kinship testing, has been adopted for the establishment of a large reference database of male European Y-chromosomal haplotypes. The extent of population stratification pertaining to this database, an issue crucial for its practical forensic application, was assessed through analysis of molecular variance (AMOVA) of the 20 regional samples included. Despite the notion of some significant haplotype frequency differences, which were found to correlate with known demographic and historic features of Europeans, AMOVA generally revealed a high level of genetic homogeneity among the populations analyzed. Owing to their high diversity, however, accurate frequency estimation is difficult for Y-STR haplotypes when realistic (i.e. moderately sized) datasets are being used. As expected, strong pair-wise and higher order allelic associations were found to exist between all markers studied, implying that haplotype frequencies cannot be estimated as products of allele frequencies. A new extrapolation method was therefore developed which treats haplotype frequencies as random variables and generates estimates of the underlying distribution functions on the basis of closely related haplotypes. This approach, termed frequency 'surveying', is based upon standard population genetics theory and can in principle be applied to any combination of markers located on the Y-chromosome or in the mitochondrial genome. Application of the method to the quality assured reference Y-STR haplotype database described herein will prove very useful for the evaluation of positive trace-donor matches in forensic casework.

Y-STR haplotyping in two Hungarian populations.

A set of seven Y-chromosomal STR loci (DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393) with the addition of the bilocal marker DYS385 was used to generate male-specific haplotype databases for two Hungarian population samples, Caucasians from the Budapest area and Romanies from Baranya county. At the locus DYS385 three types of intermediate sized alleles were detected in six males. The presence of a (GA) dinucleotide, probably due to an (AA) deletion in the second (GAAA) repeat of the polymorphic repeat region leads to an intermediate allelle 17.2. The intermediate alleles 17.-1 and 18.-1 with the consensus repeat structure of (GAAA)17 and (GAAA)18, respectively, were found to lack a T in the same (T)7 stretch located within the 3' flanking region of each allele. The forensic efficiency values for the Romany population were significantly lower than those found in the Central Hungarian and other non-isolated Causasian populations, which may imply a possible common paternal ancestry of some haplotypes in the Romany sample. With pairwise comparisons of inter-population molecular variance, the two populations analyzed here and an Italian population sample, could be clearly distinguished using the seven monoclonal Y-STRs. A sizing precision of < or = 0.14 nucleotide standard deviation was obtained with capillary electrophoresis carried out on an ABI Prism 310 Genetic Analyzer. Objective and accurate genotyping is thus possible using an internal size standard with a high density of fragments.

Fluorescence based co-amplification and automated detection of the STR loci HUMFIBRA and HUMD21S11 in a Hungarian Caucasian population sample.

Population data were generated for the STR systems HUMFIBRA and HUMD21S11 for a Hungarian Caucasian population sample residing in Baranya County, Hungary (127 unrelated individuals). The loci were coamplified using a fluorescence based PCR method and were typed automatically. For both loci 12 different alleles could be found including some variants. No deviations from Hardy-Weinberg expectations were observed. Both loci proved to be highly discriminating and valuable polymorphisms for forensic analyses.

Population genetic data on four STR loci in a Hungarian Romany population.

A population study of Hungarian Romanies was carried out on the STR loci HumLPL, HumF13B, HumFES and HumF1301. There was little evidence for association of alleles within/between the four STR systems. Allele frequency distributions were significantly different between the Romany and the previously reported Central Hungarian population databases. Population differentiation was estimated by computing F- and phi-statistics as well as frequency estimate differences of individual phenotypes for these two population samples. The results suggest that the population structure may have an effect on the interpretation of forensic DNA evidence in Hungary. Phylogenetic tree reconstruction with six populations from three major ethnic groups revealed a relatively distant genetic relationship of the Baranya Romanies with other Caucasian populations.

A population study of the STR loci HUMLPL, HUMF13B, and HUMF13A01 in Hungary.

Allele frequencies of the three STR systems HUMLPL, HUMF13B, and HUMF13A01 were determined in a Hungarian population sample of 223 unrelated Caucasian individuals. All loci met Hardy-Weinberg expectations and there was no evidence for association of alleles among the three STR loci. In addition, little evidence was found for departures from expectations of independence between any of the three STR and other previously investigated microsatellite polymorphisms.

Semi-automatic DNA profiling in a Hungarian Romany population using the STR loci HumVWFA31, HumTH01, HumTPOX, and HumCSF1PO.

A population study of Hungarian Romanies was carried out for the STR loci HumVWFA31, HumTH01, HumTPOX, and HumCSF1PO. After multiplex PCR amplification semi-automatic DNA profiling was performed using an ALF DNA sequencer. At the loci investigated there was little and no evidence for departures from Hardy-Weinberg expectations and linkage equilibrium, respectively. The allele sizing accuracy of the ALF DNA sequencer was increased to a high level (99.97% on average) by applying external and internal markers. Allele frequency distributions of the STR loci, with one exception, were significantly different between the Romany and other Hungarian population databases. On the other hand, however, only small differences in frequencies of individual phenotypes were found.

[Use of polymerase chain reaction DNA studies in Hungarian legal practice]. / Polimeráz láncreakción alapuló DNS-vizsgálatok a magyar igazságügyi gyakorlatban.

The authors survey the application of polymerase chain reaction (PCR)-based DNA polymorphisms in the Hungarian forensic practice. The combined application of the presented 17 PCR-based sequence- or length-polymorphic DNA systems to criminal cases gives the power of individualization to the hand of the forensic scientist. The joint application of these genetic markers to disputed paternity cases enables the verification of paternity for an unexcluded man with the highest legal category, namely "paternity practically proved". The investigation of sex-chromosome linked STRs and/or the analysis of mitochondrial DNA (mtDNA) D-loop region is useful in solving most of the problematic cases. The highlighted advantages of PCR over restriction fragment length polymorphism (RFLP)-based forensic DNA analyses clearly explain the overwhelming spread of PCR-based methods in the Hungarian forensic practice.

Comparative study on reused haemodialysis membranes.

Data have shown that on the internal surface of reused haemodialysis membranes complement elements of plasma proteins, cells and cellular debris from the patient's blood are deposited. The aim of our study was to identify the effect of reuse on the haemodialysis membrane surface. Original and reused haemodialysis membranes were studied by the methods of light, transmission and scanning electron microscopy, immuno-histochemistry and X-ray spectrum element analysis. With reuse the membrane surface becomes uneven, and its continuity may break. Plasma protein and cellular elements are deposited on the damaged surface following continued reuse. Presence of IgM deposists were confirmed by immunohistochemical observation. Moreover, following reuse the X-ray spectrum analysis verified structural changes in the haemodialysis tube compared to the control material; accumulated aluminium, silicon, phosphorus, sulphur and chlorine were detected on the wall of the dialysis membrane.

Hungarian population data on seven PCR-based loci.

Hungarian population data for the loci LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1, and D1S80 were generated. The genotype frequency distributions for the loci do not deviate from Hardy Weinberg expectations. Furthermore, there was little evidence for departures from expectations of independence between the loci. Using a test for homogeneity all the loci were similar between two Hungarian population samples and only the HLA-DQA1 locus was statistically different between Hungarians and US Caucasians. There generally would be little forensic differences, whether a Hungarian or a US Caucasian database was used, for estimating multiple locus profile frequencies for the seven PCR-based loci.

Hungarian population data on six STR loci--HUMVWFA31, HUMTH01, HUMCSF1PO, HUMFES/FPS, HUMTPOX, and HUMHPRTB--derived using multiplex PCR amplification and manual typing.

We present a Hungarian population study for six tetrameric short tandem repeat (STR) loci employing multiplex PCR amplification, electrophoresis of the PCR products in DNA sequencing gels and subsequent detection of allelic fragments by silver staining. The loci were HUMVWFA31, HUMTH01, HUMCSF1PO, HUMFES/ FPS, HUMTPOX, and HUMHPRTB. All loci met Hardy-Weinberg expectations in the examined Hungarian Caucasian population sample (N = 223 individuals). In addition, there was no evidence for association of alleles among the five autosomal loci HUMVWFA31, HUMTH01, HUMCSF1PO, HUMFES/FPS, and HUMTPOX.

Hungarian population data on the loci HLA-DQ alpha, LDLR, GYPA, HBGG, D7S8 and GC.

Population data studies for HLA-DQ alpha and PM loci (LDLR, GYPA, HBGG, D7S8, GC) were carried out on a Hungarian Caucasian population sample of 163 unrelated individuals. Whereas the observed PM allele frequencies were similar to those reported for Caucasians, significant differences were found for HLA-DQ alpha between the Hungarian and some Caucasian population data. All six loci meet Hardy-Weinberg expectations and there is no evidence for association between any pairs of loci.

AMPFLP analysis of the VNTR loci D1S80 and ApoB in Hungary.

Population data studies for D1S80 and ApoB were carried out on a caucasian population sample from Hungary of 229 and 222 unrelated individuals, respectively. We observed 26 different alleles for D1S80 and 13 for ApoB. The allele frequencies found are similar to those reported in the literature for European caucasians. No evidence of significant deviations from Hardy-Weinberg equilibrium were observed for both AMPFLP systems.

Hungarian population data for the STR systems TH01 and VWA.

Population data studies for TH01 and VWA were carried out on a Hungarian caucasian population sample of 244 unrelated individuals. We found 7 and 8 different alleles for TH01 and VWA, respectively. Using a high resolution/non-denaturing horizontal PAGE system no variations in the mobility of alleles were detected except for the allele 14 in VWA. The allele frequencies observed are similar to those reported in the literature for European caucasians. The genotype distributions meet Hardy-Weinberg expectations for both STR systems.