Marzena Skrzypczak-Zielinska

Familial adenomatous polyposis of the colon

Familial adenomatous polyposis (FAP) is a well-defined autosomal dominant predisposition to the development of polyposis in the colon and rectum at unusually early ages. The first symptoms of FAP are diarrhea and blood in the stool. Weight loss and weaknesses occur after the development of advanced tumour. The incidence of the FAP disorder is one per 10000 newborns. There are high levels of heterogeneity with regard to the number and timing of the occurrence of polyps. The classical form of FAP is characterized by the presence of more than 100 polyps, which appear in the second decade of life. The average time of occurrence of polyps is 15 years. The earliest symptoms of polyposis have been observed in a three-year-old child. The polyps are characterized by large potential for the development towards malignant tumour. Malignancy can occur from late childhood onwards. Attenuated adenomatous polyposis coli is characterized by a more benign course of disease in contrast to classical FAP. The occurrence of FAP is associated with mutations in the APC tumour suppressor gene, which was described in 1991. The APC gene is located on chromosome 5q21 and is involved in cell proliferation control. A recessive form of adenomatous polyposis is caused by mutations in the base excision repair gene - MUTYH gene. The MUTYH gene is involved in repairing DNA lesions as a result of oxidative DNA damage. MUTYH associated polyposis (MAP) is a predisposition to the development of polyps of the colon but the number of polyps is lower in comparison to classical FAP. The high risks of cancer observed in these two diseases make them important medical issues. Molecular studies of colonic polyposis have been performed in Poland for over fifteen years. A DNA Bank for Polish FAP patients was established at the Institute of Human Genetics in Poznan in which DNA samples from 600 FAP families have been collected.

The impact of genetic factors on response to anaesthetics

In recent years, exceptional progress has been observed in pharmacogenetics, i.e. investigations of inherited conditioning of the organisms response to drugs or xenobiotics. On the other hand, modern molecular biology techniques have been implemented, making it possible to perform studies determining the involvement of genetic factors in differing responses to agents employed in general anaesthesia. Unexpected and incorrect response of the organism to the administration of specific anaesthetics is most commonly associated with a genetic defect of the metabolic pathway of a given agent or its receptor. The majority of agents used in anaesthesia are metabolised in the liver by the cytochrome P450 superfamily enzymes (CYPs) and phase II drug-metabolising enzymes: glutathione S-transferases (GSTs), sulphotransferases (SULTs), UDP-glucuronosyltransferases (UGTs) and NAD(P)H:quinone oxidoreductase (NQO1). Propofol is presently widely used for gastrointestinal (GI) and several other procedures. Among genes associated with metabolism of the most commonly applied anaesthetics such as propofol and sevoflurane, the following ones can be mentioned: CYP2E1, CYP2B6, CYP2C9, GSTP1, UGT1A9, SULT1A1 and NQO1. Moreover, the basic mechanism of propofol action involves its interaction with an ionotropic receptor GABAA inhibiting transfer of nerve impulses. Molecular studies have shown that polymorphic changes in GABRG2 receptor gene turn out to be important in the propofol anaesthesia. Planning of optimal anaesthesia can be considerably assisted by the determination of genetic factors of prognostic value taking advantage of genotyping and making it possible to select anaesthetics and reduce risk of side effects as well as undesirable actions.

Genotype and allele frequencies of polymorphic UGT1A9 in the Polish population

The human UDP-glucuronosyltransferase 1A9 (UGT1A9) plays a central role in the metabolism of different therapeutic drugs, carcinogens and endobiotics. The UGT1A9 gene shows genetic polymorphism with frequencies significantly different in populations and ethnic groups. Many of these genetic variants are directly responsible for polymorphic drug metabolism. Three crucial alleles of UGT1A9, UGT1A9*3 (p.Met33Thr), *4 (p.Tyr242X), *5 (p.Asp256Asn) are associated with decrease or absence of enzyme activity, which intensify the risk of toxic effect during biotransformation. The goal of the present study was to discover frequencies of these genetic variations in 308 healthy individuals representing Polish population. The genotypes were determined by pyrosequencing. We demonstrated that the frequency of the variant UGT1A9*3 was 0.016, which suggests the need for detailed analysis of its effect on important drugs metabolism level in Polish population. Alleles UGT1A9*4 and UGT1A9*5 were not present in any of the subjects. So far, no studies have been conducted in which the distribution of these alleles has been determined in the Polish population.

New Analysis Method of Myotonic Dystrophy 1 Based on Quantitative Fluorescent Polymerase Chain Reaction

Molecular genetic testing of myotonic dystrophy type 1 (DM1) is based on the identification and determination of a cytosine-thymine-guanine (CTG) repeat expansion in the DMPK gene. This is usually done by Southern blot analysis-a time-consuming and very laborious technique requiring high molecular weight DNA. The aim of our study was to develop a highly sensitive, rapid, and cost-effective molecular analysis characterizing the CTG repeat region of the DMPK gene based on a two-step polymerase chain reaction (PCR) protocol. (1) For the detection of alleles of up to 100 repeats, a quantitative fluorescent (QF) amplification with primers flanking the repeat region of the DM1 locus and two reference genes (PAX2 and DHCR7) for standardization was used. By this method it was possible to identify both homozygous and heterozygous DM1 alleles. (2) Long PCR was only performed if a single wild-type allele was detected that gave a QF-PCR signal of only half intensity compared to a homozygous sample. The results obtained using combined QF and Long PCR are highly accurate compared with Southern blot analysis. We conclude that our new rapid analysis is reliable for genetic testing of DM1 patients.

The impact of genetic factors on response to glucocorticoids therapy in IBD

ABSTRACT Glucocorticosteroids (GCs) are used for many years as first-line drugs for the achievement of remission in exacerbations of inflammatory bowel disease (IBD). However, close to 20% of patients are resistant to GCs, and 40% of patients become dependent on GCs. The challenge of today’s personalized medicine is the anticipation of the steroid therapy effects even before the initiation of treatment. As several studies show, individually variable response to GCs in population has a genetic background and may depend on gene variability encoding proteins involved in the function and metabolism of GCs. To those genes belong: NR3C1 – responsible for the synthesis of GC receptor (GR); Hsp90, HSP70, STIP1, FKB5 – genes of GR protein complex; ABCB1 and IPO13 coding glycoprotein p170; and importin 13 – involved in GCs transport; IL1A, IL1B, IL2, IL4, IL8, IL10, TNF, and MIF – genes of the epithelial pro-inflammatory factors synthesis, which excessive activation causes steroid resistance as well as CYP3A4 and CYP3A5 – encoding GCs biotransformation enzymes. This work systematizes and sums up the state of current knowledge in the field of pharmacogenetics as well as expectations for the future in the realm of individualized medicine in IBD patients treated with GC drugs.

Glutathione S-transferase as a toxicity indicator in general anesthesia: genetics and biochemical function

General anesthesia may lead in patients to unexpected and adverse reactions including toxicity. Glutathione S-transferases (GSTs) are enzymes responsible for the detoxification process of anesthetic agents. Plasma and urine GST measurements are used in multiple studies as a hepatocellular integrity or renal injury indicator. The importance of GST enzyme measurements in monitoring the hepatotoxic and nephrotoxic effect in anesthetized patients is presented. The biochemical function and specific properties of GST render it a prognostic biomarker. This review demonstrates that GST can be valuable and promising toxicity indicator in patients undergoing general anesthesia.

High-resolution melting analysis of the TPMT gene: a study in the Polish population.

The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines.

Microchimerism in twins.

Introduction The aim of this paper was to report the occurrence of peripheral blood chimerism in newborns from bigeminal pregnancies. Material and methods Cord blood collected from 50 pairs of twins constituted the biological material studied. Analyses included: DNA isolation, quantitative and qualitative assessment of DNA preparations, hybridization analysis of SLS type as well as of MLS type, and analysis of microsatellite sequences with regard to polymorphisms using polymerase chain reaction. Results The presence of additional fragments of DNA in peripheral blood lymphocytes was found in four out of fifty pairs of monozygotic twins (8%) at locus D7S21 (7p22, n = 3) and locus D12S11 (12q24.3, n = 1). In these cases, the presence of additional DNA fragments was also proved by analysis of microsatellite sequence polymorphisms at loci HUMPLA2A1 (pancreatic phospholipase A-2, 12q23), HUMCYARO (cytochrome P450, 15q21.1) and HUMvWF (von Willebrand factor, 12p13). Conclusions The results of our study confirm the occurrence of chimerism in twins and constitutes the starting point for further studies aimed at determining the clinical significance of chimerism in twins both for women and fetuses.

Pharmacogenetics of Cannabinoids

Although the application of medical marijuana and cannabinoid drugs is controversial, it is a part of modern-day medicine. The list of diseases in which cannabinoids are promoted as a treatment is constantly expanding. Cases of significant improvement in patients with a very poor prognosis of glioma or epilepsy have already been described. However, the occurrence of side effects is still difficult to estimate, and the current knowledge of the therapeutic effects of cannabinoids is still insufficient. In our opinion, the answers to many questions and concerns regarding the medical use of cannabis can be provided by pharmacogenetics. Knowledge based on proteins and molecules involved in the transport, action, and metabolism of cannabinoids in the human organism leads us to predict candidate genes which variations are responsible for the presence of the therapeutic and side effects of medical marijuana and cannabinoid-based drugs. We can divide them into: receptor genes—CNR1, CNR2, TRPV1, and GPR55, transporters—ABCB1, ABCG2, SLC6A, biotransformation, biosynthesis, and bioactivation proteins encoded by CYP3A4, CYP2C19, CYP2C9, CYP2A6, CYP1A1, COMT, FAAH, COX2, ABHD6, ABHD12 genes, and also MAPK14. This review organizes the current knowledge in the context of cannabinoids pharmacogenetics according to individualized medicine and cannabinoid drugs therapy.

Bitter taste of Brassica vegetables: The role of genetic factors, receptors, isothiocyanates, glucosinolates, and flavor context

ABSTRACT It is well known that consumption of Brassica vegetables has beneficial effect on humans health. The greatest interest is focused on glucosinolates and their hydrolysis products isothiocyanates, due to their potential as cancer preventing compounds. Brassica vegetables are also rich in flavor compounds belonging to many chemical groups. The main sensory sensation related to these vegetable is their characteristic sharp and bitter taste, and unique aroma. Because of these features this group of vegetables is often rejected by consumers. Interestingly, for some people unpleasant sensations are not perceived, suggesting a potential role of inter-individual variability in bitter taste perception and sensibility. Receptors responsible for bitter sensation with the emphasis on Brassica are reviewed, as well as genetic predisposition for bitterness perception by consumers. Also the role of glucosinolates and isothiocyanates as compounds responsible for bitter taste is discussed based on data from the field of food science and molecular biology. Isothiocyanates are shown in broaded context of flavor compounds also contributing to the aroma of Brassica vegetables.