Some mistakes were made in the Supplemet issue of the journal Biochemia Medica 2006; 16(Suppl 1) (abstracts of the 5th Croatian Congress of Medical Biochemists, Poreč, Croatia). We apologize to the authors and bring corrections.
Biochemia Medica 2006;16(Suppl 1):S64
Genetic analysis of multifactorial disease: osteoporosis
Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
Osteoporosis is a disorder in which the loss of bone strength leads to fragility fractures. It is a common disease with complex pathophysiology involving both endogenous and environmental factors. Evidence from twin family studies indicates that genetic factors play an important role in the regulation of bone mineral density (BMD), which is the main phenotype of osteoporosis. The heritability of BMD has been estimated to up to 70%-80%. These phenotypes are determined by the influence of several genes and environmental factors. Therefore, osteoporosis has been considered a polygenic and multifactorial disease. Several approaches are currently being used to identify the genes that contribute to the pathogenesis of osteoporosis. At the beginning, linkage studies were mostly focused on identification of the genes responsible for monogenic bone disease. Recently, they have been oriented to identification of chromosomal regions called quantitative trait loci which genes regulate bone mass and skeletal geometry. An advantage of these family-based studies is that they are unlikely to give false positive results. However, they are less suitable for complex diseases and have a low statistical power to detect genes with modest effects on BMD. Association studies represent the main model in the genetics of osteoporosis in humans. These involve identification of polymorphisms of particular (candidate) genes and related allelic variants to BMD or bone fractures in a population-based study or case control study. Candidate genes are typically chosen on the basis of their biological effects on bone metabolism or bone cell activity. Candidate gene association studies are relatively easy to perform and can be powered to detect small effects. Disadvantages are the possibility of false positive results due to confounding factors and population stratification. There are a number of candidate genes studied in association with osteoporosis phenotype: vitamin D receptor gene, estrogen receptor genes, collagen type I a1 gene, TGF beta gene, IL-6 gene, BMP genes, LRP 5 gene, etc. The third approach in the genetics of osteoporosis refers to the genome-wide studies. They use microarray technology and are able to analyze up to 500,000 markers in one DNA sample at once. The high price is an important disadvantage of this approach. The studies in the genetics of osteoporosis have important implications for clinical practice. The genes that regulate BMD and bone fragility are potentially important as targets for new drugs, as diagnostic markers for assessment of individual risk, and as a tool for identification of treatment nonresponders in pharmacogenetic studies.
Biochemia Medica 2006;16(Suppl 1):S98
Chronic myelogenous leukemia – therapy monitoring: case report
Radić-Antolic M, Zadro R, Davidović-Mrsić S, Sertić D, Labar B
Zagreb University Hospital Center, Zagreb, Croatia
A 49-year-old female patient was diagnosed with chronic myelogenous leukemia in October 2000: hepatosplenomegaly was present along with increased white blood cell count (215x109/L) but normal red blood cell count and platelet count; the presence of the Philadelphia chromosome (translocation t(9;22)(q34;q11)) and BCR-ABL fusion gene was confirmed along with distinct leukocyte proliferation in the bone marrow. Initial therapy consisted of hydroxyurea and interferon alpha (Intron A), and the patient achieved complete hematologic response but no molecular response. Two years later, bone marrow cytogenetic analyses showed 67% of Ph positive interphase cells. The patient relapsed and imatinib 400 mg/ day was started. Six months later, complete cytogenetic remission was achieved (FISH was negative for Ph positive interphase cells) but qualitative PCR was positive for BCR-ABL. The BCR-ABL/ABL ratio was low and decreasing in the next 10 months, and partial molecular response was achieved. Imatinib was continued at 400 mg/day. Twenty-three months of the introduction of imatinib therapy, 2% of Ph positive interphase cells were found and BCR-ABL/ABL ratio increased five-fold but the patient was still in hematologic remission. During the following 6 months, imatinib therapy was discontinued, which was the probable cause of increase in the number of Ph positive interphase cells (13%) and BCR-ABL/ABL ratio (20x). The loss of complete cytogenetic remission, the increase in BCR-ABL copy number and resistance to therapy are the most common results of a mutation event in the BCR-ABL kinase domain. Increased drug concentrations can overcome some of these mutations so imatinib was increased to 600 mg/day. Four months later, evaluation showed a decrease in Ph positive interphase cells (5%) but the BCR-ABL/ABL ratio remained high. The same regimen of imatinib was continued, five months later bone marrow cytogenetics revealed 4% of Ph positive interphase cells and BCR-ABL/ABL ratio below 0.01. The last follow-up in May 2006 showed complete cytogenetic response but still detectable BCR-ABL fusion gene. This case report indicates the importance of the most sensitive part of therapy monitoring by quantitative PCR with diagnostic sensitivity between FISH analysis and qualitative PCR. This is extremely important for patients without complete molecular remission (qualitative PCR positive for BCR-ABL) where the increase in BCR-ABL copy number can predict disease progression.
Biochemia Medica 2006;16(Suppl 1):S170
Analytical validation of Seradyn-Innofluor assay for everolimus measurement
Šurina B1, Knotek M2, Cvrlje-Čolić V2, Katičić M2, Prskalo M2, Preden-Kereković V1, Flegar-Meštrić Z1
1Department of Clinical Chemistry, Merkur University Hospital, Zagreb, Croatia
2University Department of Medicine, Merkur University Hospital, Zagreb, Croatia
Individualized optimal combination of immunosuppressive therapy is one of the necessary prerequisites for the best possible outcome of transplant patients. Everolimus is a novel antiproliferative immunosuppressant used in solid organ transplantation. It is a part of immunosuppressive protocols, usually in combination with calcineurine inhibitor, or with micophenolate, with or without simultaneous steroid administration. Therapeutic drug monitoring of everolimus results in a decreased incidence of acute rejection and lower incidence of side effects in solid organ recipients treated with this immunosuppressive drug (1). The shorter half-life of everolimus facilitates achieving the target effect and a more homogeneous therapeutic scope of the drug. Therefore, for the needs of patients who had kidney transplantation performed at our Hospital, we evaluated the everolimus Seradyn-Innofluor assay, which is currently the sole such assay available on the market. The Seradyn-Innofluor assay for measuring the everolimus concentration is based on the principle of immunochemical determination with fluorophore excitation with polarized light (FPIA method). All determinations were performed on an Abbott TDx analyzer. Short analytical validation of the assay included the following: imprecision in series, day to day imprecision, inaccuracy, sensitivity, and linearity. The concentration range between 3 and 8 mg/L was set as a therapeutic scope of the assay (2). Within-series imprecision for the low, medium and high concentration range (n=10) was 7.2%, 7.9% and 3.4%, respectively. Inaccuracy for the low, medium and high concentration range, expressed on the commercial control samples, was 2.5%, 10.8% and 6.1%, respectively. Day-to-day imprecision in the samples stated above (low, medium and high, n=10) was 18.5%, 17.5% and 18.5%, respectively. Using sample dilution we demonstrated minimum sensitivity level of 2 mg/L and linearity of 40 mg/L. Satisfactory analytical assay validation was obtained. We point out a somewhat poorer day-to-day imprecision rate, KVa=18.5%, which we consider to be a limitation of the assay.