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Daria Pašalić
Editor-in-Chief
Department of Medical Chemistry, Biochemistry and Clinical Chemistry
Zagreb University School of Medicine
Šalata ul 2.
10 000 Zagreb, Croatia
Phone +385 (1) 4590 205; +385 (1) 4566 940
E-mail: dariapasalic [at] gmail [dot] com

Useful links

S05-1

Barišić K.S05-1: Biology in clinical applications: from molecular diagnostics to theragnostics. Biochemia Medica 2009;19(Suppl 1):S44.
Faculty of Pharmacy and Biochemistry, University of Zagreb, Department of Medical Biochemistry and Haematology, Zagreb, Croatia
Corresponding author:kbarisic [at] pharma [dot] hr
 
Abstract
 
The development of molecular diagnostics could be divided into three phases: first, pre-human genome project era, second post-human genome project era and the last one which could be called era of theragnostics.
The first phase includes the impact of genetics to molecular diagnostics. Genetics has become an integral part of modern medicine since Watson and Crick described their structural model of DNA. Since 1953 a lot of advances have been made in identifying mutations associated with human diseases. Further advances, including Human Genome Project, are providing new opportunities to develop better diagnostics tools.
In post-human genome era, a new field with significant impact on modern medicine has arisen and termed epigenetics. Epigenetics refers to transcriptional control that regulates gene expression and is not related to changes in DNA sequence.
Recently, a new strategy called theragnostics, combines bioinformatics, genomics, proteomics, and functional genomics as molecular biology tools essential for development of modern medicine, particularly molecular diagnostics and therapeutics.
S05-2
Borovečki F. S05-2: Novel genomic approaches in patients with neurodegenerative diseases. Biochemia Medica 2009;19(Suppl 1):S45.
Center for Functional Genomics, Clinical Hospital Center Zagreb, Zagreb Medical School University of Zagreb, Zagreb, Croatia
Corresponding author:fbor [at] mef [dot] hr
 
Abstract
 
Rapid developments int he field of genomics, enabled by the Human Genome and HapMap projects, has opened numerous possibilities in discovery of biological mechanisms involved in functional homeostasis of biological systems. New genomic technologies have enabled for the first time investigation of thousands of genes at once, which has led to new insights into complex mechanisms of transcriptional regulation. Mutant proteins which cause neurodegenerative diseases, such as huntingtin or TBP, interfere with basal and activated transcriptional machinery suggesting that alterations in gene transcription may be detectable in many tissues. Moreover, since mutant huntingtin and TBP are expressed in all tissues, including peripheral blood, we hypothesized that gene expression and promoter recruitment patterns in blood could in part reflect pathological processes observed in the brain. In order to identify differential promoter recruitment in patients with spinocerebellar ataxia 17 (SCA-17), we isolated peripheral blood mononuclear cells and performed chromatin immunoprecipitation on chip using Affymetrix Human Promoter 1.0R and Agilent Human Promoter 244K microarrays. The results of the analysis showed increased promoter recruitment by the mutant TBP on both platforms, and included genes belonging to functional groups such as energy metabolism, transcription, signaling and ubiquitin/proteasome. In order to ascertain whether increased promoter binding by mutant TBP lead to changes in transcriptional activity, we also isolated RNA from peripheral blood of SCA-17 patients and performed microarray analysis using Affymetrix Human Genome U133 2.0 PLUS genechips. Expression profiling showed that majority of the differentially expressed genes were significantly down-regulated, indicating possible transcriptional disruption by the mutant protein. In conclusion, mutant proteins, such as TBP, interfere with normal transcriptional activity in peripheral blood cells and observed alterations may reflect pathological mechanisms involved in neurodegenerative diseases.
S05-3
Canki-Klein N.S05-3: Genetic testing of single-gene disorders. Biochemia Medica 2009;19(Suppl 1):S46.
Zagreb University School of Medicine and Zagreb University Hospital Centre, Department of Neurology, Zagreb, Croatia
Corresponding author:nina [dot] canki-klain [at] zg [dot] t-com [dot] hr
 
Abstract
 
Genetic testing is the analysis of a specific gene, its product or function, or other DNA and chromosome analysis used to detect or exclude an alteration likely to be associated with a genetic disorder. In other words, the result of genetic test can confirm or rule out a suspected genetic condition or help determine a person’s chance for developing or passing on a genetic disorder. Consequently, available types of testing include: diagnostic, presymptomatic, prenatal, carrier testing and newborn screening.
Since genetic testing may open up ethical and or psychological problems it should be often accompanied by genetic counseling and informed consent. Samples for DNA testing can be any material containing cells with nucleus (usually blood, skin, amniotic fluid, chorionic villi). Protein analysis is done on a sample of tissue where the product of the gene is expressed (e.g. muscle, skin, sometimes lymphoblastoid cell line).
In principle, two different approaches to DNA analysis are available: 1) Direct or gene analysis if gene is identified, and 2) Indirect or linkage study if gene is located but still not known. The choice of analysis can vary according to the gene to be studied. It depends on how big the gene is, the type of mutation, studied population, availability of methods etc. Linkage analysis depends on availability of proband’s DNA and his/her close relatives, family relationships, paternity must be clearly established and there should not (ideally) be genetic heterogeneity, that is, more than one disease locus for the clinical phenotype.
The presentation will be illustrated by analyses done in Laboratory of Clinical Neurogenetics and Muscular Disorders, Croatian Institute for Brain Research in Zagreb University School of Medicine.
S05-4
Sertić J.S05-4:Molecular diagnosis of polygenic diseases. Biochemia Medica 2009;19(Suppl 1):S47-S48.
Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine and Clinical Hospital Center, Zagreb, Croatia
Corresponding author:jadranka [dot] sertic [at] kbc-zagreb [dot] hr
 
Abstract
 
Background: During past twenty-five years major advances occurred in the genetics of polygenic disorders, such as cardiocerebrovascular (involving hypertension, hyperlipidemia, obesity) and malignant disease. The mutations that commonly occur in some genes have been directly related to defects of plasma lipid transport and predisposition to premature coronary artery disease. A common mutation in Apo CIII has in some cases been shown to associate with a predisposition to premature coronary artery disease and myocardial infarction. Genetic variants of another lipid transport protein, apolipoprotein E, have been shown to relate to both disorders of plasma cholesterol transport and the development of premature coronary artery disease. The genetics of other multifactorial disorders (e.g. diabetes mellitus, venous thromboebolism, obesity, Alzheimer’s disease) has also been partially clarified in the present decade, and has provided an abundance of new genetic markers.
The correlation of gene variants with biological variables or clinical assessments has not been well understood, especially among apparently healthy subjects.
Human obesity is a multifactorial syndrome influenced by both environmental and genetic factors. Among gene variants found to be involved in body weight regulation and the development of obesity, particular attention has been paid to polymorphisms in the genes related to adipogenesis, energy expenditure, and insulin resistance. Peroxisome proliferator-activated receptors PPARs are members of the nuclear hormone receptor subfamily of ligand-dependent transcription factors. The isoform PPARG2 is mainly expressed in adipose tissue where it modulates the expression of target genes involved in adipocyte differentiation, insulin sensitivity and inflammatory processes. We explored the association of some genetic polymorphisms of: PPARG2 (Pro12Ala); adiponectin (ADIPOQ -11391G>A and 11377C>G); IL-6 (-174G>C); TCF7L2 (rs7903146); eNOS (-786T>C); estrogen receptor (ESR1alfa-TA); APOE; ACE (I/D); MTHFR (-677C>T); LPL (PvuII+/-), with clinical variables: gender, age, BMI, and biological variables: triglycerides, cholesterol, HDL, LDL, CRP, homocysteine, glucose in 105 healthy young subjects (age range 20-35 yrs) of Croatian origin.
Methods: The genotyping of PPARG2, IL-6, ACE, LPL, eNOS was performed by PCR-RFLP, TCF7L2, APOE, MTHFR, ADIPOQ, by real-time PCR and ESR1alpha by capillary electrophoresis. Associations of alleles, genotypes and haplotypes with biological variables were performed using UNPHASED-3.0.10. Sex, age, BMI was used as covariates.
Results: BMI was increased (> 25 kg/m2) in 22% of subjects. Increased cholesterol values (> 5.0 mmol/L) were found in 23% of subjects, LDL (> 3.0 mmol/L) in 23%, triglycerides (> 1.7 mmol/L) in 11% of subjects. We found statistically significant differences in subjects’ weight (P = 0.015), BMI (P = 0.023), and hip/waist ratio (P = 0.015) in regard to their diet type; subjects with Mediterranean diet had the lowest values compared to those on continental and mixed diet. Significant associations were found for: LPL genetic polymorphic variant and abdominal obesity (P = 0.018), APOE4 variant and hypercholesterolemia (P = 0.002), and ESR1-L allele and hypercholesterolemia (P = 0.023). No association of other genetic variants with biological variable has been found so far in our study.
Conclusions: ESR1, LPL, and APO E genetic polymorphic variants could represent predictive genetic risk markers for lipid status and obesity in young healthy subjects. Mediterranean type of diet is also an important positive factor in abdominal obesity.
S05-5
Grahovac B.S05-5: Molecular HLA-DNA typing. Biochemia Medica 2009;19(Suppl 1):S48-S49.
Clinical Institute of Laboratory Diagnostic, Department of pathology, School of Medicine and Clinical Hospital Center Rijeka, University of Rijeka, Rijeka, Croatia
Corresponding author:blazenka [dot] grahovac [at] medri [dot] hr
 
Abstract
 
The Human Leukocyte Antigen (HLA) region is the human equivalent of the Major Histocompatibility Complex (MHC) located on the short arm of chromosome 6 (6p21.3). HLA genes comprise a cluster of more than 200 loci encoding integral membrane proteins that bind antigenic peptides and present them to the T-cell receptors of either the CD8 (class I HLA molecules) or CD4 (class II HLA molecules) thymus-derived lymphocytes. The functional HLA genes are highly polymorphic and most of the variability is concentrated at specific sites (the peptide-binding region) in exons 2 and 3 (class I genes) or exon 2 only (class II genes). Because of its clinical significance (the mechanism how HLA polymorphisms influence foreign tissue to be rejected by the host and by which certain alleles and haplotypes are associated with progression to infectious diseases and susceptibility to a wide range of autoimmune and other chronic, non-infectious diseases), large sets of HLA population data have been accumulated. Nucleotide sequences for more than 3,528 different alleles of these genes have been determined by April 2009.
A variety of techniques have been applied for HLA tissue typing. For many years, HLA polymorphisms were typed by serological responses to HLA antigens. However, the advent of recombinant DNA technology has revealed identification of genetic differences among the HLA loci directly. Today, clinical laboratories use several different types of HLA DNA typing methods. In general, HLA DNA can be typed either by hybridizing labeled, sequence – specific oligonucleotide probes to HLA loci amplified with generic primers by the polymerase chain reaction (PCR), or by using allele specific primers for PCR amplification directly to detect HLA polymorphisms. Each HLA DNA typing technique has its specific features, and clinical laboratory personnel must choose the most appropriate test for their needs based on cost, average turnaround time, required resolution, and number of average samples per day. For those laboratories with access to sequencing technology, it may be the most sensitive, specific and cost-effective option to sequence certain HLA loci directly.