Optimization of the Y831C mutation detection in human DNA polymerase gamma by allelic discrimination assay.

Many well-defined mutations in the gene for the catalytic subunit of polymerase gamma (POLG1) have been found to be associated with disease, whereas the status of several mutations remains unresolved due to the conflicting reports on their frequencies in populations of healthy individuals. Here, we have developed a highly sensitive, real-time allelic discrimination assay enabling detection of the Y831C mutation in the POLG1 gene. The Y831C mutation is present in the Polish population at a frequency of 2.25%. The new assay is well suited to both extensive population studies and molecular diagnostics of POLG1.

Allelic and haplotypic frequencies at 11 Y-STR loci in Buryats from South-East Siberia.

We have obtained Y-STR haplotypes in 12 loci (DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439) from 215 Buryat males. We have found that one haplotype (15-11,18-13-28-23-10-11-14-14-10-12) comprises more than 30% of Y chromosomes in this population while another haplotype (14-11,13-14-30-23-10-14-14-14-10-10) comprises additional 14% of chromosomes. The population under study seems to be very homogenous as far as Y chromosome is regarded and the most frequent haplotype seems to be the modal haplotype for Buryats.

Contrasting patterns of Y-chromosome variation in South Siberian populations from Baikal and Altai-Sayan regions.

In order to investigate the genetic history of autochthonous South Siberian populations and to estimate the contribution of distinct patrilineages to their gene pools, we have analyzed 17 Y-chromosomal binary markers (YAP, RPS4Y(711), SRY-8299, M89, M201, M52, M170, 12f2, M9, M20, 92R7, SRY-1532, DYS199, M173, M17, Tat, and LLY22 g) in a total sample of 1,358 males from 14 ethnic groups of Siberia (Altaians-Kizhi, Teleuts, Shors, Tuvinians, Todjins, Tofalars, Sojots, Khakassians, Buryats, Evenks), Central/Eastern Asia (Mongolians and Koreans) and Eastern Europe (Kalmyks and Russians). Based on both, the distribution pattern of Y-chromosomal haplogroups and results on AMOVA analysis we observed the statistically significant genetic differentiation between the populations of Baikal and Altai-Sayan regions. We suggest that these regional differences can be best explained by different contribution of Central/Eastern Asian and Eastern European paternal lineages into gene pools of modern South Siberians. The population of the Baikal region demonstrates the prevalence of Central/Eastern Asian lineages, whereas in the populations of Altai and Sayan regions the highest paternal contribution resulted from Eastern European descent is revealed. Yet, our data on Y-chromosome STRs variation demonstrate the clear differences between the South Siberian and Eastern European R1a1-lineages with the evolutionary ages compatible with divergence time between these two regional groups.

[Analysis of the importance of Helicobacter pylori strains in children with gastric and duodenal mucosal inflammatory changes]. / Analiza wystepowania i wplywu zakazenia szczepami CagA(+) Helicobacter pylori na zmiany zapalne blony sluzowej zoladka i dwunastnicy u dzieci.

UNLABELLED: The role of Helicobacter pylori (H. pylori) infection in etiopathogenesis of gastritis and/or duodenitis is well recognized and proven. H. pylori infection also concerns children and youth. It was proven that CagA(+) strains of H. pylori cause inflammatory changes and can lead to ulcer disease of ventriculum and/or duodenum. AIM: The purpose of the study was to determine the prevalence of urease A and cytotoxicity genes in children with diagnosed H. pylori infection and concomitant gastritis and/or duodenitis as well as to determine the kind and stadium of inflammatory changes in relation to particular strains. MATERIAL AND METHODS: The study was performed in 76 patients aged from 3 to 20 years (average age 12.6), who were qualified for endoscopy of the upper GI tract because of recurrent abdominal pain complaints. The H. pylori infection and accompanying gastritis and/or duodenitis were diagnosed according to the Sydney classification. RESULTS: In 57/76 (75%) of diagnosed patients the H. pylori infection was proven with the PCR method (ureA (+)). The following strains were found: CagA(+) in 19 (33% infected subjects). Gastritis and/or duodenitis occurred in all subjects infected with CagA (+) strains and in 54/57 of those with H. pylori infection ureA (+). In subjects without infection, gastritis and/or duodenitis was found in 12 persons, and this result was significantly lower. CONCLUSIONS: In diagnosis it is important to confirm H. pylori presence by CagA gene typing because it relates to the inflammatory changes of the stomach and/or duodenum.

Low variability of the POLG (CAG)n repeat in north Eurasian populations.

We investigated the frequency of different repeat-length alleles of the trinucleotide CAG microsatellite repeat in the coding sequence of the nuclear gene for the catalytic subunit of mitochondrial DNA polymerase gamma (POLG) in 12 ethnic groups from northern Eurasia. The population sample consisted of 1,330 individuals from 3 large geographic areas: Europe, Southwest Asia, and Siberia/East Asia. We found that the 10-repeat allele of the POLG gene is the most frequent in all analyzed populations, with a frequency of 88-96%. The heterozygosity level ranges from 22% in Europe to 13.6% in Southwest Asia with the lowest value of 7.4% in Siberia/East Asia. The present study provides evidence of clinal distribution of POLG gene heterozygosity in North Eurasian populations. In general, we found an extremely low variability of the trinucleotide CAG microsatellite repeat, suggesting that purifying selection acts against deleterious alleles, although low mutability of the repeated region cannot be ruled out.

Differentiation of mitochondrial DNA and Y chromosomes in Russian populations.

The genetic composition of the Russian population was investigated by analyzing both mitochondrial DNA (mtDNA) and Y-chromosome loci polymorphisms that allow for the different components of a population gene pool to be studied, depending on the mode of DNA marker inheritance. mtDNA sequence variation was examined by using hypervariable segment I (HVSI) sequencing and restriction analysis of the haplogroup-specific sites in 325 individuals representing 5 Russian populations from the European part of Russia. The Y-chromosome variation was investigated in 338 individuals from 8 Russian populations (including 5 populations analyzed for mtDNA variation) using 12 binary markers. For both uniparental systems most of the observed haplogroups fell into major West Eurasian haplogroups (97.9% and 99.7% for mtDNA and Y-chromosome haplogroups, respectively). Multidimensional scaling analysis based on pairwise F(ST) values between mtDNA HVSI sequences in Russians compared to other European populations revealed a considerable heterogeneity of Russian populations; populations from the southern and western parts of Russia are separated from eastern and northern populations. Meanwhile, the multidimensional scaling analysis based on Y-chromosome haplogroup F(ST) values demonstrates that the Russian gene pool is close to central-eastern European populations, with a much higher similarity to the Baltic and Finno-Ugric male pools from northern European Russia. This discrepancy in the depth of penetration of mtDNA and Y-chromosome lineages characteristic for the most southwestern Russian populations into the east and north of eastern Europe appears to indicate that Russian colonization of the northeastern territories might have been accomplished mainly by males rather than by females.

The bovine tyrosine hydroxylase gene associates in vitro with the nuclear matrix by its first intron sequence.

Recently we have shown that in vitro binding of the proximal part of the human tyrosine hydroxylase gene to the nuclear matrix is correlated with its transcriptional activity. The strongest binding potential was predicted by computing for the first intron sequence (Lenartowski & Goc, 2002, Neurosci Lett.; 330: 151-154). In this study a 16 kb fragment of the bovine genomic DNA containing the tyrosine hydroxylase gene was investigated for its affinity to the nuclear matrix. Only a 950 bp fragment encoding the distal part of the first intron, second exon and a few nucleotides of the second intron bound to the nuclear matrix. The binding was independent of the tissue-specific tyrosine hydroxylase gene activation. The fragment was subcloned and sequenced. Computer search pointed to one potential intronic matrix attachment region with two AP1-like sites embedded in the sequence. We conclude that even if the position of the matrix binding region is conserved among the tyrosine hydroxylase genes in mammals, its tissue specificity and/or function is not preserved or is achieved by different mechanisms.

High levels of mitochondrial DNA heteroplasmy in single hair roots: reanalysis and revision.

The present study demonstrates a reinvestigation of the mitochondrial DNA sequence heteroplasmy, which was previously found by the use of nested polymerase chain reaction (PCR) technique in single hairs of 13 individuals. The direct PCR approach was used for the amplification of mitochondrial DNA and a phylogenetic analysis was applied to both data sets for the verification of the authenticity of sequences. The comparative analysis of the sequencing results obtained from the same hair DNA extracts - but using two different techniques - shows that direct mitochondrial DNA amplification results in a considerably lower number of mixed positions. The majority of the confirmed heteroplasmic variants preferentially occurs in mitochondrial DNA hypervariable sites (mutational hotspots). However, the pattern of heteroplasmic mutations observed in four extracts after nested PCR significantly differs from the pattern of natural mutations. Some of these rare polymorphisms should be revised as inconsistent with phylogenetic expectations. The results of the present study contribute to the earlier reports by indicating that phylogenetic analysis is an effective tool in a posteriori quality check of mitochondrial DNA data.

Analysis of microsatellite instability and loss of heterozygosity in breast cancer with the use of a well characterized multiplex system.

Analysis of microsatellite instability (MI) and loss of heterozygosity (LOH) is recommended for screening patients with sporadic and hereditary malignancies. This study shows an application of a fluorescent hexaplex PCR system for microsatellite typing on A.L.F. DNA Sequencer (Pharmacia Biotech). This technique detects changes in microsatellites providing a time-efficient, reliable and accurate method for MI and LOH analyses. The Fragment Manager software was used for automated size calculation and quantitation of DNA fragments, enabling rapid and precise measurement of allelic ratios. We examined 70 breast cancer and 70 control DNA specimens, classified all the patterns of microsatellite alterations, and set up MI and LOH assessment criteria for the automated multiplex fluorescent method.