Use of direct sequencing for detection of mutations in the BCR-ABL kinase domain in Slovak patients with chronic myeloid leukemia.

The presence of BCR-ABL oncogene mutations in patients with chronic myeloid leukemia (CML) may be responsible for the failure of tyrosine kinase inhibitor (TKI) treatment. The aim of the study was to evaluate the frequency of BCR-ABL gene mutations in patients with CML treated with tyrosine kinase inhibitors. Our lab received 64 samples (34 women, 30 men) from patients with CML who failed or had suboptimal response to TKI treatment. The mutation analysis was performed in 61 patients with CML, 3 patients could not be tested because of inadequate RNA quality. An 866 base pair fragment containing the ABL kinase domain was amplified in a seminested RT (reverse transcriptase)-PCR and then sequenced using Applied Biosystems BigDye Terminator chemistry with two pairs of primers. We analyzed 61 patients with CML, 11 mutations were detected in 13 (21%) patients and SNP (single nucleotide polymorphism) in 6 patients (10%). In addition to 9 point mutations (G250E / F317L, F359V, L387M, Y253H, M388L, M244V, T315I, D276G), 35 bp insertion between exons 8 and 9 and deletion exon 7 were detected. Our results demonstrate that direct sequencing is suitable for routine clinical monitoring patients with CML and may be useful for optimizing therapy.

Novel approaches in evaluation of pathogenicity of single-base exonic germline changes involving the mismatch repair genes MLH1 and MSH2 in diagnostics of Lynch syndrome.

Germline defects in the DNA mismatch repair genes MLH1 and MSH2 are the major cause of hereditary nonpolyposis colon cancer (HNPCC), also called Lynch syndrome. Detection of inherited pathogenic change in their DNA sequence in HNPCC families allows for identification of asymptomatic individuals who require appropriate medical surveillance. However, evaluation of clinical significance of identified DNA alteration is not always straight-forward and some changes maybe classified incorrectly depending on the method used. The aim of this review is to summarize rationale, practice and pitfalls in the characterization of substitutions localized in the exons and outline new experimental and in silico approaches used to determine mutation consequence. Our survey of variants identified in MLH1 and MSH2 genes which were confirmed to cause splicing defect but often appear characterized as missense, nonsense or silent mutations in various databases and publications as well as a list of true missense mutations may serve as a valuable aid for laboratories providing HNPCC diagnosis.

Birth control in antiquity.

The question of birth rate control in Antiquity clashes against problems in the sense that the majority of ancient authors when discussing this matter expressed themselves in a very unclear way; they did not distinguish exactly between contraceptives and abortifacients and sometimes even interchanged them mutually. The ancient Greek doctors studied long and precisely this area of gynaecology and especially Hippocrates and his school contributed a lot to study of abortions and abortifacients. Contraceptive theories and methods proclaimed by Greek and Roman doctors meant a significant progress in development of medical science. The opinions of doctors of Hippocrates group, Sorano of Efes and others, confirm their importance for medical practice by their wide and frequent occurrence in medical writings. Some methods of contraception defended by Greek and Roman doctors were very effective and many pieces of information became basis for modern contraceptive means, others certainly did not achieve the desired effects.

The role of clinical criteria, genetic and epigenetic alterations in Lynch-syndrome diagnosis.

Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) represents 1-3% of all diagnosed colorectal cancers (CRCs). This study aimed to evaluate the benefit of clinical criteria and several molecular assays for diagnosis of this syndrome. We examined tumors of 104 unrelated clinically characterized colorectal cancer patients for causal mismatch repair (MMR) deficiency by several methods: microsatellite instability (MSI) and loss of heterozygosity (LOH) presence, MMR protein absence, hypermethylation of MLH1 promoter and germline mutation presence. Twenty-five (24%) patients developed CRCs with a high level of MSI (MSI-H). Almost all (96%) had at least one affected relative, while this simple criterion was satisfied in only 22% (17/79) of individuals with low level MSI or stable cancers (MSI-L, MSS). Using strict Amsterdam criteria, the relative proportion of complying individuals in both sets of patients (MSI-H vs. MSI-L and MSS) decreased to 68% and 9%, respectively. The right-sided tumors were located in 54% of MSI-H persons when compared to 14% of cancers found in MSI-L or MSS patients. In 16 MSI positive patients with identified germline mutation by DNA sequencing, the gene localization of mutation could be indicated beforehand by LOH and/or immunohistochemistry (IHC) in four (25%) and 14 cases (88%), respectively. The IHC findings in MSI-H cancers with methylation in distal or both regions of MLH1 promoter have not confirmed the epigenetic silencing of the MLH1 gene. None of the patients with MSIL or MSS tumors was a carrier of the MLH1 del616 mutation, despite seven of them meeting Amsterdam criteria. The effective screening algorithm of Lynch-syndrome-suspected patients consists of evaluation of Bethesda or Revised Bethesda Guidelines fulfilling simultaneous MSI, LOH and IHC analyses before DNA sequencing. Variable methylation background in MLH1 promoter does not affect gene silencing and its role in Lynch-syndrome tumorigenesis is insignificant.

STK11/LKB1 germline mutations in the first Peutz-Jeghers syndrome patients identified in Slovakia.

Peutz-Jeghers syndrome (PJS) is characterized by number of hamartomatous polyps in the gastrointestinal tract and by mucocutaneous hypermelanocytic lesions at different sites. Older patients have an increased risk of the cancers of small intestine, stomach, pancreas, colon, esophagus, ovary, testis, uterus, breast and lung. In majority of PJS cases, the germline mutations in serine/threonine kinase STK11/LKB1 gene were found to be associated with disease. Here we report the results of a first mutational screen of STK11/LKB1 in PJS patients characterized in Slovak population. The first patient with unusual carcinoma of duodenum was a sporadic case and carried c.842delC change residing in a mutational C6 repeat hotspot. Neither the polyp nor the tumor of the patient displayed the loss of heterozygosity at the site of mutation suggesting different mechanism involved in the formation of polyp and tumor in this case. The second patient belonged to a three-generation family with typical PJS features but not cancers. Interestingly, the patient displayed concomitant occurrence of adenomatous and hamartomatous polyps. Molecular analysis revealed an IVS2+1A>G mutation that alters the second intron 5' splice site and was shown to lead to aberrant splicing mediated by the U12-dependent spliceosome. The same mutation was present in the 9 affected members of the family but in none of their normal relatives. We also observed novel c. IVS2+61G>A unclassified variant, and recurrent IVS2+24G>T and 3UTR+129C>T polymorphisms. Based on the achieved results, we could offer predictive genetic testing and counseling to other members of the patient's families.

Novel and recurrent germline alterations in the MLH1 and MSH2 genes identified in hereditary nonpolyposis colorectal cancer patients in Slovakia.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes, predominantly MSH2 and MLH1. Mutation carriers develop cancers in the colorectum, endometrium, ovary, stomach, small intestine and the upper urinary tract. We describe here the results of a mutational analysis of 11 unrelated HNPCC patients by direct genomic sequencing of MLH1 and MSH2. The alterations found include 7 novel changes and 4 different pathogenic mutations described previously in Poland, Moldavia, Finland, Germany, France and USA. Four novel pathogenic mutations in the MLH1 gene include two frameshift mutations (c.1150delG and c.1210_1211delCT), one missense mutation (c.793C>A) and one intron-exon border mutation (c.546- 2A>C). The last change resulted in the skipping of exon 7, as shown by sequencing of RT-PCR products. The only novel MSH2 pathogenic change was a nonsense mutation c.1129C>T. The novel intronic change c.381-41A>G in MLH1 was found in a patient carrying a previously-described mutation in the MSH2 gene. Interestingly, two unrelated patients carried also a novel change in the promoter region of MLH1 in one of the CpG islands (c.-269C>G). However, this alteration does not abrogate transcription, as shown by RT-PCR analysis. In summary, most (approximately 80%) pathogenic germline mutations detected in the studied group of patients by direct genomic sequencing of MLH1 and MSH2 were located in the MLH1 gene. These and previous data indicate that the majority of germline point mutations and small deletions/insertions in HNPCC families in Slovakia affect the MLH1 locus.

Application of word-formation models in medical terms fixation.

In their article the authors present goals of Latin teaching at medical faculties in Slovakia. They analyze in more details word-formation and structure of one-word medical terms from the point of view of their model teaching/learning and fixation. Presentation of the medical terms in models proved to be very effective for adult learners because they provide exact and easy-to-memorize scheme mechanisms that can be analogically applied in the production/manipulation and fixation of a whole range of medical terms. In conclusion several examples--excercises are presented to illustrate their use in teaching practice. (Fig. 1, Ref. 7.).

Hippocrates and his principles of medical ethics.

"The Father of Medicine", as Antiquity called Hippocrates has left rich medical and ethical heritage for us. His heritage--the collection of treatises Corpus Hippocraticum, from 5th and 4th centuries BC, comprise not only general medical prescriptions, descriptions of diseases, diagnoses, dietary recommendations etc., but also his opinion on professional ethics of a physician. The Hippocratic Oath, taken by ancient and medieval doctors, requires high ethical standards from medical doctors. Its principles are important in professional and ethical education of medical doctors even today. (Ref. 4.).

Hippocrates' humoral pathology in nowaday's reflections.

Ancient medicine integrated three components: experience (empirical observation), religion/magic and speculations of natural philosophers. The Greek medicine out-achieved medicine of other ancient nations in starting to investigate the true causes of health and diseases and thus laying foundations for the diagnosis, prognosis and treatment. Hippocrates, the most famous physician of the ancient times, made a synthesis of existing philosophical opinions from the point of view of a physician. His 58 writings were preserved in the collection "Corpus Hippocraticum". The most relevant writing in it--"Peri fyseos anthropu" (On the nature of man)--is ascribed to Hippocrates' son-in-law Polybos to whom we are grateful for the ancient humoral-pathological theory. In explaining human organism and its processes the author integrated ancient teachings on 4 basic humors (humoral theory), elements and qualities with observations of manifestations of health and disease. Normal condition (health) was defined as balance between the body fluids (eukrasia) and external environment. If this balance is disturbed, the result is dyskrasia, i.e. disease studied by pathology. According to Hippocrates disease causes can be understood only through empirical study. A man has a power to overcome disease, but to achieve it the right diet is necessary to keep harmony in body fluids of an organism. The role of a physician was just to support the nature. "Prognosis", another writing included in "Corpus Hippocraticum", reflects Hippocrates' understanding of prognosis as a necessary development of diagnosis based on past knowledge (anamnesis) and present observation. Ideas of Hippocrates and his medical school are still valuable and inspiring especially for today's very sophisticated medicine--concept of fighting diseases by natural means: maintaining healthy lifestyle and harmony within the organism, or an effort for perfect understanding of human creature and for humanization of medicine. (Fig. 3, Ref. 14.)

[Eponyms in medical terminology]. / Eponymá v lekárskej terminológii.

Use of eponyms and their frequency in anatomical and clinical medical terminologies are compared. In the standard anatomical terminology, use of eponyms in the macroscopic anatomical terms is not allowed. In the clinical terminology, on the other hand, the use of eponyms is very frequent. In conclusion the author presents advantages and disadvantages of the eponym use. (Ref. 7.)