New clustering methods for population comparison on paternal lineages.

The goal of this study is to show two new clustering and visualising techniques developed to find the most typical clusters of 18-dimensional Y chromosomal haplogroup frequency distributions of 90 Western Eurasian populations. The first technique called "self-organizing cloud (SOC)" is a vector-based self-learning method derived from the Self Organising Map and non-metric Multidimensional Scaling algorithms. The second technique is a new probabilistic method called the "maximal relation probability" (MRP) algorithm, based on a probability function having its local maximal values just in the condensation centres of the input data. This function is calculated immediately from the distance matrix of the data and can be interpreted as the probability that a given element of the database has a real genetic relation with at least one of the remaining elements. We tested these two new methods by comparing their results to both each other and the k-medoids algorithm. By means of these new algorithms, we determined 10 clusters of populations based on the similarity of haplogroup composition. The results obtained represented a genetically, geographically and historically well-interpretable picture of 10 genetic clusters of populations mirroring the early spread of populations from the Fertile Crescent to the Caucasus, Central Asia, Arabia and Southeast Europe. The results show that a parallel clustering of populations using SOC and MRP methods can be an efficient tool for studying the demographic history of populations sharing common genetic footprints.

Population genetic study in Hungarian canine populations using forensically informative STR loci.

Allele frequencies for 10 short tandem repeats (STRs) were determined using the StockMarks® Dog Genotyping Kit (Applied Biosystems) from a pool of 668 unrelated dogs, consisting of 79 different breeds or breed variants from the Hungarian canine population. For the comparative statistical analysis, four pure bred, one mixed group - all individuals except from the four breeds - and considering to unequal representation of breeds the group of all pooled individuals ("All breeds") were distinguished. The forensically informative values - Hardy-Weinberg equilibrium (HWE), observed heterozygosity (H(obs)), polymorphism information content (PIC), power of discrimination (PD), power of paternity exclusion (PE), linkage disequilibrium (LD) and fixation index (F) were determined. The Hungarian pure bred dog populations could be distinguished by comparing the allele frequency values using G-statistics and calculating the F(ST) indices with pair-wise comparisons of inter-population molecular variance (AMOVA). The results showed that these 10 loci can be adequate for individual identification in forensic cases even in relatively inbred dog populations.

Migration rates and genetic structure of two Hungarian ethnic groups in Transylvania, Romania.

Transylvania's ethnic mosaic is composed of Romanians, German Saxons and Hungarians. The ethnic groups of the Hungarian minority that settled in Romania show differences in dialects, customs and religious affiliations. In this study entire mtDNA control region sequences from 360 individuals of Hungarian ethnicity from two populations (the Csángó and the Székely), settled in the historical region of Transylvania in Romania, were generated and analyzed following high quality sequencing standards. Phylogenetic analyses were used for haplogroup determination, quasi-median network analyses were applied for the visualization of character conflicts, and median joining reconstructions were used for depicting haplotype structures. Affiliation of haplotypes to major west Eurasian haplogroups was confirmed using coding region SNPs. Gene flow between the two populations was low and biased towards a higher migration rate from the Csángó to the Székely than vice versa. Phylogeographic analyses revealed effects of genetic isolation within the Csángó population, which is, in its genetic structure, clearly different from the Székely population. The pronounced genetic divergence between the two populations is in sharp contrast to the expectation of high genetic similarity due to the close geographic proximity of their native homelands. The population data will be incorporated in the EMPOP database (http://www.empop.org).

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.

Resolution of parentage in dogs by examination of microsatellites after death of putative sire: case report.

A case of disputed paternity in dogs is reported. DNA examinations were carried out from hair samples of the individuals several months after the death of the putative sire. Ten short tandem repeat (STR) loci were analysed by fluorescence-labelled multiplex PCR using ABI PRISM 310 Genetic Analyser. Based on the results the candidate sire was included in the pedigree records as the biological sire. In spite of the genetic homogeneity of pedigree dogs due to inbreeding, canine microsatellites can provide an adequate basis for assigning paternity in pure breeds.

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.

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.

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.

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.