Gene therapy - once just a dream, now a reality.

Gene therapy is gradually becoming a mainstream treatment modality and is no longer the preserve of large university departments whose laboratories master nucleic acid analytical procedures and whose clinical teams manage its administration. It was originally designed for genetic diseases that, because of their prevalence, were a group known as rare diseases. Gene therapy has so far been applied in children to act before the disease development. These new treatments have also begun to be applied for common diseases such as metabolic disorders (e. g. diabetes) and even for those that are increasingly affecting us, such as various malignancies and diseases of the central nervous system (e. g. Alzheimer's disease). The targets targeted by GT are genes, where pathogenic alterations in the form of pathogenic variants (formerly mutations) induce phenotypic disorders, and our aim is either to knock them out of function (e. g. haemoglobinopathies) or to replace them with genes with normal function, which we introduce into the genome using one of the appropriate vectors, such as viruses or liposomes. The process of GT can take place directly inside the patient's body (in vivo) or outside the body on isolated cells (ex vivo), which are usually stem cells (iPSCs, induced pluripotent stem cell). After treatment, these cells are returned to the patient's body to fulfil their "destiny". In a broader sense, GT can target the product of gene transcription, which is the messenger RNA, or the end product of gene function, such as functional proteins (eg. cystic fibrosis). Any of these approaches have been used successfully in various diseases, depending on their availability, which is determined, among other things, by the costs associated with GT or the accessibility of the target tissue. Ultimately, it is not only the validation of the efficacy and safety of GT, but also economic reasons that determine why GT has been slow to develop and is mostly undertaken only by large and wealthy institutions. Another decisive factor is that from initial experimental work through clinical trials, the whole process of its development normally takes up to a decade.

Artificial intelligence and modern information and communication technologies entering medicine.

Many new technologies based on computer technologies which are very successful in industry spread over the medicine and became integral part of all its disciplines. Artificial intelligence opened new possibilities for managing and solving many problems in both - theoretical and practical health care. The capability of these new technologies to extract tiny interactions of different items has been appreciated especially in treatment complex diseases. They are capable to analyze not only enormous amounts of data (big data) in an extremely short time but also these processes of analyses are easily improved by machine itself (machine learning). Examples of AI application in several medical disciplines and itinerary for Electronic Health Records adoption in the Czech health care are listed.

Enlarging the gene-geography of Europe and the Mediterranean area to STR loci of common forensic use: longitudinal and latitudinal frequency gradients.

BACKGROUND: Tetranucleotide Short Tandem Repeats (STRs) for human identification and common use in forensic cases have recently been used to address the population genetics of the North-Eastern Mediterranean area. However, to gain confidence in the inferences made using STRs, this kind of analysis should be challenged with changes in three main aspects of the data, i.e. the sizes of the samples, their distance across space and the genetic background from which they are drawn. AIM: To test the resilience of the gradients previously detected in the North-Eastern Mediterranean to the enlargement of the surveyed area and population set, using revised data. SUBJECTS AND METHODS: STR genotype profiles were obtained from a publicly available database (PopAffilietor databank) and a dataset was assembled including >7000 subjects from the Arabian Peninsula to Scandinavia, genotyped at eight loci. Spatial principal component analysis (sPCA) was applied and the frequency maps of the nine alleles which contributed most strongly to sPC1 were examined in detail. RESULTS: By far the greatest part of diversity was summarised by a single spatial principal component (sPC1), oriented along a SouthEast-to-NorthWest axis. The alleles with the top 5% squared loadings were TH01(9.3), D19S433(14), TH01(6), D19S433(15.2), FGA(20), FGA(24), D3S1358(14), FGA(21) and D2S1338(19). These results confirm a clinal pattern over the whole range for at least four loci (TH01, D19S433, FGA, D3S1358). CONCLUSIONS: Four of the eight STR loci (or even alleles) considered here can reproducibly capture continental arrangements of diversity. This would, in principle, allow for the exploitation of forensic data to clarify important aspects in the formation of local gene pools.

[Control (editing) of the genome within reach, or already in our hands?] / Ovládání (úpravy) genomu na dosah anebo jiz v nasich rukou?

Although different genome editing tools have been around for decades, the recent emergence of cheap, quick, and accessible CRISPR/Cas9 technology has led to a revolution in this field. The technique has the potential to transform medicine from curative into preventive using a gene therapy. An application of genome editing has proven to be effective for both genetic and non-genetic (e.g. infectious) diseases. However, cancer and rare diseases treatment is at the forefront of interest. Concurrently, the legal and ethical frameworks should be discussed, especially as the technology moves towards a modification of the germ cells or embryos. In addition to a precise molecular genetic diagnosis and choosing the best gene therapy approach for a particular individual, an attention should also be paid to the impacts on the entire human population and the next generations. In this review, we summarize the most important applications of CRISPR/Cas9 technology in the field of medicine. Some interesting results from recent years are presented in the context of used approaches and importance for the future developments in medicine. Finally, ethical and legal conditions in relation to different gene editing applications are discussed.

[Contribution of instruments and devices to medicine personalization]. / Prístrojový prístup k personalizaci medicíny (vysetrení u pacienta, Point of Care Testing)*

In this paper we are concerned not about "large and heavy", and very expensive medical instruments with high productivity, which are able to analyze enormous amounts of samples and are suitable to examine high number of patients - populations. We deal with much "smaller and lighter" devices, with limited range of targets, but effective for personal use. We observe something like a flood of these medgadgets, tools for individual testing. They can operate if placed both at the patients bed, on human body surface as well as inside the body and due to the progress in telecommunication technologies they can send information originating from their sensors via mobile phones or tablets, both to the patient and also to his physician (health care institution). The use of these devices has become available practically for all medical branches.

Genetic structure of pastoral and farmer populations in the African Sahel.

Traditional pastoralists survive in few places in the world. They can still be encountered in the African Sahel, where annual alternations of dry and wet seasons force them to continual mobility. Little is known about the genetic structure of these populations. We present here the population distribution of 312 hypervariable segment I mitochondrial DNA (mtDNA) and 364 Y-short tandem repeat haplotypes in both farmer and pastoralist groups from the Lake Chad Basin and the West African Sahel. We show that the majority of pastoral populations (represented in the African Sahel by the Fulani nomads) fail to show significant departure from neutrality for mtDNA as evidenced by Fu's Fs statistics and exhibit lower levels of intrapopulation diversity measures for mtDNA when contrasted with farmers. These differences were not observed for the Y chromosome. Furthermore, analyses of molecular variance and population distributions of the mtDNA haplotypes show more heterogeneity in the sedentary groups than in the pastoralists. On the other hand, pastoralists retain a signature of a wide phylogenetic distance contributing to their male gene pool, whereas in at least some of the farmer populations, a founder effect and/or drift might have led to the presence of a single major lineage. Interestingly, these observations are in contrast with those recorded in Central Asia, where similar comparisons of farmer and pastoral groups have recently been carried out. We can conclude that in Africa, there have been no substantial mating exchanges between the Fulani pastoralists coming to the Lake Chad Basin from the West African Sahel and their farmer neighbors. At the same time, we suggest that the emergence of pastoralism might be an earlier and/or a demographically more important event than the introduction of sedentary agriculture, at least in this part of Africa.

Deregulation of gene expression induced by environmental tobacco smoke exposure in pregnancy.

INTRODUCTION: Environmental tobacco smoke (ETS) exposure in pregnant women may have detrimental effects such as spontaneous abortion, lower birth weight, stillbirth, and reduced infant lung function. To extend our knowledge on the molecular effects of tobacco smoke exposure in pregnancy, we analyzed transcriptome alterations in passive smokers (PS) and compared them with those in active smokers (AS). METHODS: Using Illumina Expression Beadchips with 24,526 transcript probes, gene expression patterns were assayed in placentas from PS (N = 25) exposed to ETS throughout pregnancy and nonexposed (NS) counterparts (N = 34) and in cord blood cells from their newborns. ETS exposure was evaluated by questionnaire disclosure and cotinine measurement in maternal and cord blood. RESULTS: A total of 158 genes were significantly deregulated in the placentas of PS compared with NS. These genes were associated with the extracellular matrix, apoptosis, placental function, blood clotting, response to stress, and lipid metabolism. Cord blood of the newborns of PS displayed differential expression of 114 genes encoding mainly adhesion molecules and regulators of immunologic response. A comparison of the affected pathways between PS and AS indicated that ETS exposure and active smoking in pregnancy partly employ the same molecular mechanisms. CONCLUSIONS: This study demonstrates that even low dose exposure to ETS during pregnancy leads to significant deregulation of transcription in placental and fetal cells. These data suggest that the effect of ETS on the fetus is primarily indirect, mediated via deregulation of placental functions.

[Prediction in medicine--genome contra envirome]. / Predikce v medicíne--genom contra envirom.

Human phenotype is governed by its genotype--a set of genetic information materialized in DNA. Using traditional terminology we speak about a little more than 20 thousands genes that differ in strength to become realized and their effect is modified by a large number of other genes. The result originates from firmly established programmes we obtained from our ancestors. Development and activity of such molecules selected for maintenance, copying and transfer of information i.e. nucleic acids can be followed back to the very origin of the life. Nevertheless the final result is achieved not only by confrontation of the original information with other genetic information but largely also by external influences--environment. Though we are relatively successful in understanding what we have inherited from our parents, our knowledge of environmental factors and their effects on formation of the phenotype is still limited. From this point of view medical prediction has always to be very cautious and interpretations at the probability level must be done by a very experienced and responsible professional.

[Principles of dealing with human DNA and genotyping information]. / Principy pro zacházení s lidským genetickým materiálem a genotypizacní informací

Subject of human DNA analysis is so far unsatisfactorily dealt within the Czech legislature. Unfortunately, it is not that easy to set up transparent and unambiguous rules comprehensively covering the whole subject. In this paper, basic principles of dealing with DNA are covered using the process view (sampling, DNA extraction, genotyping, archiving, and data mining), thus unifying medicinal, individualizing, genealogical, explorative, and phenotyping points of view. DNA law should consist of requirements for procedural steps of handling DNA and its analysis, for persons (both natural and legal), and for laboratories testing DNA. At the same time, processes to control the law-abiding and risk minimizing should be set up. The well prepared DNA law would allow to fulfil a potential of DNA analysis: to help substantially the healthcare, the judiciary, and other users of genotyping results. In the best scenario, quality of genotyping results and public confidence in genetic testing will be raised, risk of misuse minimized, human rights well balanced, market purged, and genotyping service rationalized. We believe that it is a worthwhile task.

Hypertrophic cardiomyopathy: from mutation to functional analysis of defective protein.

AIM: To analyze the genesis of hypertrophic cardiomyopathy on a large cohort of patients from molecular genetics point of view and perform the functional analysis of the 3D molecular model of defective myosin-7 protein in silico. METHODS: The study enrolled 153 patients with diagnosed hypertrophic cardiomyopathy from different parts of the Czech Republic. DNA samples were analyzed for mutations in exons 21 and 22 of the MYH7 gene, which have been associated with high mutation clustering. The 3D model of human myosin-7 was built using the x-ray structure of nucleotide-free scallop myosin S1 as the structural template. We performed de novo structure prediction of mutant and wild type peptides spanning the 769-788 amino acids region of the myosin-7 protein. RESULTS: The Arg870His and Asp778Val amino acid alterations were found in 2 unrelated patients with a severe form of hypertrophic cardiomyopathy. The Asp778Val variation was chosen for subsequent 3D molecular modeling in silico. The mutation of the Asp by Val not only changes the character of the interaction pattern with other amino acids or ions but Val, being a small hydrophobic amino acid, can also completely change the stability of the region. CONCLUSION: Mutation location in the MYH7 gene and changes in amino acid composition may have a crucial negative impact on the outcome of the disease in patients with hypertrophic cardiomyopathy. In addition, a mutation that changes the charge of the amino acid is more likely to affect protein function than a conservative mutation.

[National Database of Genotypes--ethical and legal issues]. / Národní databáze genotypu--etické a právní aspekty.

National Database of Genotypes--ethical and legal issues The aim of the project National Database of Genotypes is to outline structure and rules for the database operation collecting information about genotypes of individual persons. The database should be used entirely for health care. Its purpose is to enable physicians to gain quick and easy access to the information about persons requiring specialized care due to their genetic constitution. In the future, another introduction of new genetic tests into the clinical practice can be expected thus the database of genotypes facilitates substantial financial savings by exclusion of duplicates of the expensive genetic testing. Ethical questions connected with the creating and functioning of such database concern mainly privacy protection, confidentiality of personal sensitive data, protection of database from misuse, consent with participation and public interests. Due to necessity of correct interpretation by qualified professional (= clinical geneticist), particular categorization of genetic data within the database is discussed. The function of proposed database has to be governed in concordance with the Czech legislation together with solving ethical problems.

[Postgenomic era, what comes after?]. / Postgenomová era a co príjde po ní?

Though we start to speak about postgenomic era, the genomic era has not been finished yet and the structure, function and variability of our genome is being still intensively studied and these studies bring us continually new scientific information--more than we are able to digest. The classical genetics utilized phenotype observation for discovering the function of genetic information and proceeded to the molecular basis represented by nucleic acids. Determination of the nucleotide sequence of the human genome is the top outcome of the effort. At present, the function, regulatory pathways and genome modifications have become principal targets of our research. If we compare variability, it increases in the direction from human genome to transcriptome and to proteom reaching the highest level in phenome. Differences concern not only quantity, but also quality with the exception of genome which is relatively stable and "we hand over to our children what we have inherited from our parents"--all other levels undergo dynamic changes, and from this point of view are much less stable and under continuous influence of environment. To understand enviromental factors shaping our phenome, a long-term monitoring of our living functions will be necessary and an instrumental approach has to be looked for.

Differential gene expression in umbilical cord blood and maternal peripheral blood.

OBJECTIVES: Umbilical cord blood (UCB) has become a useful alternative source of hematopoietic stem cells for clinical and research applications. UCB represents neonatal blood and differs from adult blood in many aspects, displaying different cell composition and various features of cellular immaturity. To understand molecular basis of phenotypic differences between neonatal and adult blood, we studied variations in transcriptome of UCB and maternal peripheral blood (PB). METHODS: Using Illumina microarrays, we determined gene expression profiles of UCB and PB samples obtained from 30 mothers giving birth to living baby. RESULTS: Out of 20,589 tested genes, 424 genes were down-regulated and 417 genes were up-regulated in UCB compared with PB. Reduced expression of many immunity-related pathways (e.g. TLR pathway, Jak-STAT pathway, cytokine-cytokine receptor interaction) in neonatal blood cells may contribute to the poor response to antigens, increasing susceptibility to infections at the time of disappearance of protective maternal antibodies. On the other hand, overexpression of erythropoiesis-related genes (glycophorins, fetal hemoglobins, enzymes catalysing heme synthesis and erythrocyte differentiation) in UCB probably enforces red cell production in newborns. CONCLUSIONS: Our study demonstrates that neonatal and maternal bloods show specific gene expression profiles, likely reflecting differences in phenotypes of immunologically immature and fully evolved hematopoietic cells.

Expression analysis of PCNA gene in chronic myelogenous leukemia--combined application of siRNA silencing and expression arrays.

Imatinib metylase is the first choice treatment for BCR/ABL positive chronic myelogenous leukemia (CML). However, as some CML patients develop resistance to imatinib therapy, there is a significant interest in development of alternative treatment strategies, such as identifying targets other than BCR/ABL that may participate in CML. Previously, we demonstrated strong PCNA up-regulation in CML patients. To further study its role in CML pathogenesis, we performed silencing of PCNA expression followed by array experiments. PCNA inhibition led to down-regulation of CDK1, CDK4, PLK1, ERK3, JNK1, STAT5, and several inhibitors of apoptosis (DAXX, Mdm2, survivin). The following genes were up-regulated: CDK inhibitors p21 and p19-INK4D, pro-apoptotic FAST kinase, fibronectin, etc. However, as PCNA affects cell growth in naturally proliferating cells as well as in cancerous cells, it seems to act a secondary role relating to proliferation activity of leukemic cells.

Bmi-1 over-expression plays a secondary role in chronic myeloid leukemia transformation.

It has been demonstrated that over-expression of Bmi-1 occurs in a variety of cancers, including several types of leukemia. This gene plays a key role in the self-renewal of stem cells. Leukemic cells lacking Bmi-1 underwent proliferation arrest and showed signs of differentiation and apoptosis. These findings led to the proposal of Bmi-1 as a potential target for therapeutic intervention in cancer. In this study, we investigated the role of Bmi-1 in chronic myeloid leukemia (CML). Using qRT-PCR, we demonstrated a significantly increased level of Bmi-1 transcript in CML cells. Using array analysis, we determined the deregulation of several genes after Bmi-1 silencing. Proapoptotic genes BAD and TRADD, and CASP8, p16-INK4, BRCA2, Notch4 and Wnt-8B were elevated. PLK1, SOD1, E2F-3, two retinoblastoma binding proteins (RBQ1 and RBBP4) and HDGF were reduced after Bmi-1 inhibition. Additionally, we tested the impact of Bmi-1 siRNA on CML cell growth; however, there was no apparent change after Bmi-1 suppression. Despite the fact that Bmi-1 deregulation occurs in CML and its expression is connected to several oncogenic processes, Bmi-1 seems to play a secondary role in CML transformation.

Report of an international survey of molecular genetic testing laboratories.

OBJECTIVE: To collect data on the practices of molecular genetic testing (MGT) laboratories for the development of national and international policies for quality assurance (QA). METHODS: A web-based survey of MGT laboratory directors (n = 827; response rate 63%) in 18 countries on 3 continents. QA and reporting indices were developed and calculated for each responding laboratory. RESULTS: Laboratory setting varied among and within countries, as did qualifications of the directors. Respondents in every country indicated that their laboratory receives specimens from outside their national borders (64%, n = 529). Pair-wise comparisons of the QA index revealed a significant association with the director having formal training in molecular genetics (p < 0.005), affiliation with a genetics unit (p = 0.003), accreditation of the laboratory (p < 0.005) and participation in proficiency testing (p < 0.005). Research labs had a lower mean report score compared to all other settings (p < 0.05) as did laboratories accessioning <150 samples per year. CONCLUSION: MGT is provided under widely varying conditions and regulatory frameworks. The data provided here may be a useful guide for policy action at both governmental and professional levels.

Spatially Explicit Models to Investigate Geographic Patterns in the Distribution of Forensic STRs: Application to the North-Eastern Mediterranean.

Human forensic STRs used for individual identification have been reported to have little power for inter-population analyses. Several methods have been developed which incorporate information on the spatial distribution of individuals to arrive at a description of the arrangement of diversity. We genotyped at 16 forensic STRs a large population sample obtained from many locations in Italy, Greece and Turkey, i.e. three countries crucial to the understanding of discontinuities at the European/Asian junction and the genetic legacy of ancient migrations, but seldom represented together in previous studies. Using spatial PCA on the full dataset, we detected patterns of population affinities in the area. Additionally, we devised objective criteria to reduce the overall complexity into reduced datasets. Independent spatially explicit methods applied to these latter datasets converged in showing that the extraction of information on long- to medium-range geographical trends and structuring from the overall diversity is possible. All analyses returned the picture of a background clinal variation, with regional discontinuities captured by each of the reduced datasets. Several aspects of our results are confirmed on external STR datasets and replicate those of genome-wide SNP typings. High levels of gene flow were inferred within the main continental areas by coalescent simulations. These results are promising from a microevolutionary perspective, in view of the fast pace at which forensic data are being accumulated for many locales. It is foreseeable that this will allow the exploitation of an invaluable genotypic resource, assembled for other (forensic) purposes, to clarify important aspects in the formation of local gene pools.

Array-based analysis of gene expression in childhood acute lymphoblastic leukemia.

Gene expression profiles of 10 children with acute lymphoblastic leukemia (ALL) were detected using cDNA arrays. Total RNAs were isolated from peripheral blood leukocytes of the patients at diagnosis. For detection of expression profiles we used Atlas Human Cancer cDNA Arrays (Clontech) with 588 genes. Although the study revealed variability of gene expression in many genes, we identified a number of genes with the same expression changes (up-regulation: PCNA, ERCC1; down-regulation: jun-B, BCL-2 related protein A1, CRAF-1, PBP) in most examined patients. Our objective was to identify genes that were differentially expressed in ALL and might contribute to development (and characterization) of the disease.

Gene expression profiling in chronic myeloid leukemia patients treated with hydroxyurea.

Using array technology that allows the simultaneous detection of gene expression of hundreds of genes, four patients with chronic myeloid leukemia (CML) were investigated at diagnosis and after starting administration of hydroxyurea. To detect the gene expression of peripheral blood mononuclears and granulocytes Human Cancer cDNA Array (CLONTECH) with 588 gene probes was used. Gene expression mononuclear and granulocyte profiles of patients at diagnosis were compared with the control profiles. The significant expression changes observed in most patients seemed to be important. Increased expression of c-jun N-terminal kinase 2 (JNK2), integrin alpha E, MMP-8, MMP-9 was detected in both fractions of most patients. In some samples PCNA, HDGF, MAPK p38, CD59 increased expressions were found. Significant down-regulation of expression in patients was detected in genes CDK4 inhibitor A, PURA, notch1 in mononuclears; STAT2, STAT5, RAR-alpha, MCL-1, junB, caspase 4 in granulocytes; CDK6, GADD153, ERBB-3, cadherin 5 in both fractions. Expression profiles detected in patients at diagnosis did not differ markedly from those after one-week treatment with hydroxyurea. Only in a few genes were significant changes after hydroxyurea administration observed and inter-individual expression differences were rather common.