Tadeusz Dobosz

Characteristics and frequency of germline mutations at microsatellite loci from the human Y chromosome, as revealed by direct observation in father/son pairs.

A number of applications of analysis of human Y-chromosome microsatellite loci to human evolution and forensic science require reliable estimates of the mutation rate and knowledge of the mutational mechanism. We therefore screened a total of 4,999 meioses from father/son pairs with confirmed paternity (probability >/=99. 9%) at 15 Y-chromosomal microsatellite loci and identified 14 mutations. The locus-specific mutation-rate estimates were 0-8. 58x10-3, and the average mutation rate estimates were 3.17x10-3 (95% confidence interval [CI] 1.89-4.94x10-3) across 8 tetranucleotide microsatellites and 2.80x10-3 (95% CI 1.72-4.27x10-3) across all 15 Y-chromosomal microsatellites studied. Our data show a mutational bias toward length increase, on the basis of observation of more repeat gains than losses (10:4). The data are in almost complete agreement with the stepwise-mutation model, with 13 single-repeat changes and 1 double-repeat change. Sequence analysis revealed that all mutations occurred in uninterrupted homogenous arrays of >/=11 repeats. We conclude that mutation rates and characteristics of human Y-chromosomal microsatellites are consistent with those of autosomal microsatellites. This indicates that the general mutational mechanism of microsatellites is independent of recombination.

Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications.

Nonrecombining Y-chromosomal microsatellites (Y-STRs) are widely used to infer population histories, discover genealogical relationships, and identify males for criminal justice purposes. Although a key requirement for their application is reliable mutability knowledge, empirical data are only available for a small number of Y-STRs thus far. To rectify this, we analyzed a large number of 186 Y-STR markers in nearly 2000 DNA-confirmed father-son pairs, covering an overall number of 352,999 meiotic transfers. Following confirmation by DNA sequence analysis, the retrieved mutation data were modeled via a Bayesian approach, resulting in mutation rates from 3.78 × 10(-4) (95% credible interval [CI], 1.38 × 10(-5) - 2.02 × 10(-3)) to 7.44 × 10(-2) (95% CI, 6.51 × 10(-2) - 9.09 × 10(-2)) per marker per generation. With the 924 mutations at 120 Y-STR markers, a nonsignificant excess of repeat losses versus gains (1.16:1), as well as a strong and significant excess of single-repeat versus multirepeat changes (25.23:1), was observed. Although the total repeat number influenced Y-STR locus mutability most strongly, repeat complexity, the length in base pairs of the repeated motif, and the fathers age also contributed to Y-STR mutability. To exemplify how to practically utilize this knowledge, we analyzed the 13 most mutable Y-STRs in an independent sample set and empirically proved their suitability for distinguishing close and distantly related males. This finding is expected to revolutionize Y-chromosomal applications in forensic biology, from previous male lineage differentiation toward future male individual identification.

Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR® Yfiler® PCR amplification kit

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpFlSTR® Yfiler® polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730–1,764 DNA-confirmed father–son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son’s birth) of fathers with mutations was with 34.40 (±11.63) years higher than that of fathers without ones at 30.32 (±10.22) years, a difference that is highly statistically significant (p < 0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father’s age on a statistically significant level (α = 0.0294, 2.5% quantile = 0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father–son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses.

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier’s algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

Homogeneity and distinctiveness of Polish paternal lineages revealed by Y chromosome microsatellite haplotype analysis

Abstract. Different regional populations from Poland were studied in order to assess the genetic heterogeneity within Poland, investigate the genetic relationships with other European populations and provide a population-specific reference database for anthropological and forensic studies. Nine Y-chromosomal microsatellites were analysed in a total of 919 unrelated males from six regions of Poland and in 1,273 male individuals from nine other European populations. AMOVA revealed that all of the molecular variation in the Polish dataset is due to variation within populations, and no variation was detected among populations of different regions of Poland. However, in the non-Polish European dataset 9.3% (P<0.0001) of the total variation was due to differences among populations. Consequently, differences in RST-values between all possible pairs of Polish populations were not statistically significant, whereas significant differences were observed in nearly all comparisons of Polish and non-Polish European populations. Phylogenetic analyses demonstrated tight clustering of Polish populations separated from non-Polish groups. Population clustering based on Y-STR haplotypes generally correlates well with the geography and history of the region. Thus, our data are consistent with the assumption of homogeneity of present-day paternal lineages within Poland and their distinctiveness from other parts of Europe, at least in respect to their Y-STR haplotypes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00439-002-0728-0.

Association studies of CTLA-4, CD28, and ICOS gene polymorphisms with B-cell chronic lymphocytic leukemia in the Polish population

Abnormal expression of the costimulatory molecules cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD28, and inducible co-stimulator (ICOS) leads to disturbances of immune response and an increased risk of cancer. An extended study was undertaken to evaluate the association among the polymorphisms CTLA-4c.49A>G, CTLA-4g.319C>T, CTLA-4g.*642AT(8_33), CD28c.17+3T>C, and ICOSc.1554+4GT(8_15) and susceptibility to B-cell chronic lymphocytic leukemia (B-CLL) in the Polish population. The study revealed increased frequency of the CTLA-4g.319C>T [T] allele and the CTLA-4g.319C>T [T] phenotype in B-CLL patients compared with healthy controls (p = 0.003, odds ratio [OR] = 1.73; and p = 0.009, OR = 1.74, respectively). The presence of the CD28c.17+3T>C [C] allele and the CD28c.17+3T>C [C] phenotype increased the OR of B-CLL to 1.59 (p = 0.007) and 1.74 (p = 0.007), respectively. Either CTLA-4g.319C>T or CD28c.17+3T>C was associated with time to Rai stage progression. The distributions of the alleles and genotypes of the ICOS gene significantly differed between patients and controls (p = 0.0009 and p = 0.006, respectively). Individuals possessing short alleles were 2.02 times more prone to B-CLL than others (p = 0.001), whereas carriers of long alleles were protected from B-CLL (p = 0.02, OR = 0.62). The haplotype association study and multivariate analysis confirmed the association of CTLA-4g.319C>T and ICOSc.1554+4GT(8_15) gene polymorphisms with B-CLL. The polymorphic sites CTLA-4c.49A>G and CTLA-4g.*642AT(8_33) did not correlate with B-CLL. Our results are the first in the literature to report that gene polymorphism of the costimulatory molecules CTLA-4, CD28, and ICOS contributes to susceptibility to B-CLL.

The history of Slavs inferred from complete mitochondrial genome sequences.

To shed more light on the processes leading to crystallization of a Slavic identity, we investigated variability of complete mitochondrial genomes belonging to haplogroups H5 and H6 (63 mtDNA genomes) from the populations of Eastern and Western Slavs, including new samples of Poles, Ukrainians and Czechs presented here. Molecular dating implies formation of H5 approximately 11.5–16 thousand years ago (kya) in the areas of southern Europe. Within ancient haplogroup H6, dated at around 15–28 kya, there is a subhaplogroup H6c, which probably survived the last glaciation in Europe and has undergone expansion only 3–4 kya, together with the ancestors of some European groups, including the Slavs, because H6c has been detected in Czechs, Poles and Slovaks. Detailed analysis of complete mtDNAs allowed us to identify a number of lineages that seem specific for Central and Eastern Europe (H5a1f, H5a2, H5a1r, H5a1s, H5b4, H5e1a, H5u1, some subbranches of H5a1a and H6a1a9). Some of them could possibly be traced back to at least ∼4 kya, which indicates that some of the ancestors of todays Slavs (Poles, Czechs, Slovaks, Ukrainians and Russians) inhabited areas of Central and Eastern Europe much earlier than it was estimated on the basis of archaeological and historical data. We also sequenced entire mitochondrial genomes of several non-European lineages (A, C, D, G, L) found in contemporary populations of Poland and Ukraine. The analysis of these haplogroups confirms the presence of Siberian (C5c1, A8a1) and Ashkenazi-specific (L2a1l2a) mtDNA lineages in Slavic populations. Moreover, we were able to pinpoint some lineages which could possibly reflect the relatively recent contacts of Slavs with nomadic Altaic peoples (C4a1a, G2a, D5a2a1a1).

Progression of multiple sclerosis is associated with exon 1 CTLA-4 gene polymorphism.

Objectives – Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system which is widely believed to have a T‐cell‐mediated etiology. The cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) antigen molecule plays a key role in the downregulation of T‐cell responses. To examine the genetic association of the CTLA‐4 gene locus with MS, we analyzed an exon 1 (A49G) transition.

Genetic data from Y chromosome STR and SNP loci in Ukrainian population

We have tested a sample of 154 unrelated males from Lviv region (Ukraine) for 11 Y-chromosomal single nucleotide polymorphisms (SNPs) and 17 Y-chromosomal STR loci (DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, YGATA_H4.1). Haplotype and haplogroup diversity values were calculated for the population under study. Genetic distances (R(ST)) to 9 other Slavic populations were calculated based on 12 Y-STR loci. Haplotype frequencies and MDS plots were constructed based on genetic distances. Haplogroup frequency patterns revealed in Ukraine are similar to those characteristic of other European populations. However, it also allowed for identification a specific genetic component in Ukrainian sample which seems to originate from areas dwelled by Western Slavs, i.e. subhaplogroup R1a1a7, at frequency of 13.65%. Analysis of R(ST) distances and AMOVA revealed high level of heterogeneity between Slavic populations inhabiting the south and north part of Europe, determined geographically rather than by linguistic factors. It has also been found a closer similarity (in the values of R(ST)) between Ukrainian and Slovak populations than between Ukrainians and other Slavic population samples.

Proinflammatory and atherogenic activity of monocytes in Type 2 diabetes

UNLABELLED Cytokines secreted by the monocyte-macrophage system play a key role in the progression of atherosclerotic lesions in Type 2 diabetes. The objectives of this study were to assess the influence of cytokine gene expression in monocytes from patients with Type 2 diabetes on direct markers of endothelial injury with regard to clinically manifest atherosclerosis. METHODS Monocytes from 58 patients with Type 2 diabetes and from 22 age-matched healthy volunteers of a control group were isolated in order to assess expression of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-6, IL-8 and IL-10 cytokines (RTPCR, Applied Biosystems). Thrombomodulin concentration was determined using a Diagnostica Stago Immunoenzymatic assay, and circulating endothelial cell numbers were assayed using immunofluorescence studies with CLB-HEC19 antibodies. RESULTS In 28 patients, TNFalpha expression in monocytes was observed. In these patients, as compared to those with undetectable levels of this cytokines expression, higher hemoglobin A(1c) (P=.012) and thrombomodulin (P=.005) concentrations were found. IL-8 expression was determined in 36 patients. Higher expression of TNFalpha (P=.048) and IL-8 (P=.049) was detected in patients with peripheral arterial disease in contrast to those free from this complication. CONCLUSION TNFalpha and IL-8 play a significant role in the proatherogenic activity of monocytes in Type 2 diabetes. The TNFalpha-connected activity of monocytes may directly determine endothelial dysfunction and injury. The location of atherosclerosis should be taken into account in the assessment of the proinflammatory activity of peripheral blood monocytes.