Ryszard Słomski

APC gene mutations causing familial adenomatous polyposis in Polish patients

Familial adenomatous polyposis (FAP) is a well-known hereditary condition characterised by alimentary system tumours. Tens to thousands of polyps occur in the colon and rectum of the patients. There is a high heterogeneity with regard to the number and time of the occurrence of polyps. The occurrence of FAP is associated with mutations in theAPC tumour suppressor gene, which was described in 1991. Since then, many studies have been done to analyse the distribution of mutations in individual populations and to determine the function of the gene and a diagnostic approach to FAP. Here theAPC gene was studied with respect to the occurrence of small mutations and large rearrangements in 300 unrelated Polish FAP families. Ninety-seven mutations were identified in 164 families. Out of these mutations, 80 were small mutations, including 58 small mutations that were first identified in the Polish population (42 novel and 16 described previously). An increased frequency of mutation c.3927_3931delAAAGA was observed in 10% of the Polish group. Seventeen large rearrangements were found in 29 families. Out of those rearrangements, 8 repeat rearrangements occurred in 20 families. A problem in fast molecular diagnostics of FAP is a high heterogeneity of mutations in theAPC gene. It seems that a multiplex ligation-dependent probe amplification test and searching for small mutations by the use of screening methods at the 5’ end of exon 15 and exons 14, 9, 11, 13, 5, and 3, help to improve the molecular diagnostics of FAP in Polish patients.

Generation of Transgenic Rabbits by the Novel Technique of Chimeric Somatic Cell Cloning

Abstract A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

Discrimination between recurrent mutation and identity by descent : application to point mutations in exon 11 of the cystic fibrosis (CFTR) gene

SummaryA total of 75 non-ΔF508 chromosomes from 59 German cystic fibrosis patients was screened for mutations in exon 11 of the cystic fibrosis (CFTR) gene. These Caucasian patients were found to possess an identical haplotype background for two common mutations (G551D, R553X) constistent with their being identical by descent. However, a different R553X associated haplotype found in American black patients was suggestive of recurrent mutation, a postulate supported by the location of the R553X alteration in a hypermutable CpG dinucleotide. Likelihood estimates for recurrent mutation and identity by descent were compared and strongly supported the hypothesis of recurrent R553X mutation. The ability to distinguish between these two alternatives provides an indication of whether or not the search for mutations should be restricted to chromosomes with similar haplotypes.

Paternity testing with oligonucleotide multilocus probe (CAC)5/(GTG)5 : a multicenter study

The statistical analysis is reported of 256 paternity cases referred to seven different German laboratories for multilocus DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5 and restriction enzyme HinfI. All parameters characteristic of multilocus DNA fingerprints were found to differ significantly between the contributing centres: the number of analyzed gel positions, the number of bands scored per individual, the probability of occurrence of a band at a particular position, and the band-sharing probabilities between the mother and both child and alleged father. Despite these differences, paternity cases could be divided clearly into two distinct subgroups on the basis of (i) offspring bands that could not be assigned to either the mother or the alleged father and (ii) the extent of band-sharing between child and alleged father. This partitioning, which is likely to correspond to true and false paternity, confirms previous findings for other multilocus probes. A goodness-of-fit test on the normalized number of bands scored per individual revealed no systematic deviations from commonly adopted analytical models regarding electrophoretic bands as independent entities. Log10-likelihood ratios of paternity vs. non-paternity were calculated utilizing one of these models, and a clear-cut partitioning was again obtained which coincides with that mentioned before. Only one case could not be decided unambiguously, and was either due to two independent mutations or to a close relative of the alleged father being the true father.

Risk factors in abdominal aortic aneurysm and in Polish population aortoiliac occlusive disease and differences between them [corrected].

Abdominal aortic aneurysm (AAA) and aortoiliac occlusive disease (AIOD) are multifactorial vascular disorders caused by complex genetic and environmental factors. The purpose of this study was to define risk factors of AAA and AIOD in the Polish population and indicate differences between diseases.

Genetically Modified Pigs as Organ Donors for Xenotransplantation

The growing shortage of available organs is a major problem in transplantology. Thus, new and alternative sources of organs need to be found. One promising solution could be xenotransplantation, i.e., the use of animal cells, tissues and organs. The domestic pig is the optimum donor for such transplants. However, xenogeneic transplantation from pigs to humans involves high immune incompatibility and a complex rejection process. The rapid development of genetic engineering techniques enables genome modifications in pigs that reduce the cross-species immune barrier.

Recurrent APC gene mutations in Polish FAP families

The molecular diagnostics of genetically conditioned disorders is based on the identification of the mutations in the predisposing genes. Hereditary cancer disorders of the gastrointestinal tracts are caused by mutations of the tumour suppressor genes or the DNA repair genes. Occurrence of recurrent mutation allows improvement of molecular diagnostics. The mutation spectrum in the genes causing hereditary forms of colorectal cancers in the Polish population was previously described. In the present work an estimation of the frequency of the recurrent mutations of the APC gene was performed. Eight types of mutations occurred in 19.4% of our FAP families and these constitute 43% of all Polish diagnosed families.

Is G→T substitution in the sequence of CAG repeats within the androgen receptor gene associated with aggressive behaviour in the red foxVulpes vulpes?

Studies were carried out on one of the largest European red foxVulpes vulpes (Linnaeus, 1758) farms in Śniaty and Batorówka (Poland). A test created by Nowicki and Przysiecki (NP) was used to describe behaviour of the animals. The results of the NP behaviour test showed 4 types of behaviour in foxes: aggressive, curious, indifferent and apprehensive. While analyzing a fragment of exon 1 in the androgen receptor gene in 184 individuals, four alleles were found, ie 10, 10T, 12 and 13. The most frequent allele was allele 10, both in males and females (65.85 and 57.39%, respectively). The next in order of frequency were allele 10T (24.39 and 31.25%), 13 (7.32 and 9.09%) and 12 (2.44 and 2.27%, respectively). On the basis of further analysis an association was shown between behaviour of the red fox and its genotype. In aggressive females allele 10 was found significantly more frequently (76%) than in curious females (57%). While analyzing the genotypes of aggressive females it was shown that there were 15 individuals with genotype 10/10 (15.56%), 11 heterozygotes (9.87%) and only 1 individual with genotype 10T/10T (1.56%). In curious females the distribution of these genotypes was 15 (13.71%), 18 (20.57%) and 9 (7.71%), respectively. Although the result of Pearson Χ2 analysis was not significant (Χ2,p=0.0793), the Armitage’s chi-squared test for trend showed a significant difference Χ2,p=0.0305). This results may suggest that the androgen receptor gene may be suitable in studies on psychological traits.

Ten new ATM alterations in Polish patients with ataxia‐telangiectasia

Inherited biallelic mutations of the ATM gene are responsible for the development of ataxia telangiectasia (AT). The objective of the present study was to conduct molecular analysis of the ATM gene in a cohort of 24 Polish patients with ataxia‐telangiectasia with aim being to provide an updated mutational spectrum in Polish AT patients. As a result of molecular analysis, the status of recurrent mutation was confirmed and ten new ATM variants were detected. Application of MLPA analysis allowed the detection of large genomic deletion. Previously, this type of mutation had never been seen in our population. Finally, in silico analysis was carried out for newly detected ATM alterations. In addition, functional analysis was performed to evaluate the effects of intronic variants: c.3402+30_3402+32delATC.

The Secondary Structure Model of Mouse U1 snRNA as Determined from the Results of Pb-Induced Hydrolysis

All eukaryotic cells contain a group of small nuclear RNAs, designated UsnRNAs, that function in mRJSfA and rRNA processing as ribonucleoprotein snRNPs (Busch et al., 1982). The primary structure of Ul snRNA, the most abundant UsnRNA in animal cells, is known for several closely related as well as distant species, including human, rat, mouse, chicken, frog, fruit fly, peas and beans (Reddy, 1988). The molecules, depending on the organism, are 162–166 nucleotides long and they are thought to form the same secondary structure (Reddy and Busch, 1988) which seems to be also preserved in intact Ul snRNP particles, that are the essential components of spliceosomes (Chabot and Steitz, 1987).