Daria Pašalić
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

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Genotyping of 13 α-thalassaemia point mutations using primer extension reaction and dipstick assay
Petropoulou M
National and Kapodistrian University of Athens, Department of Chemistry, Athens, Greece
Corresponding author: mpetrop [at] biol [dot] uoa [dot] gr
Background: Alpha-thalassaemia is inherited as an autosomal recessive disorder characterized by a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. Reduced or absent a-globin synthesis, mainly caused by deletions of one or both a-globin genes and by point mutations, leads to a-thalassemia (a-thal). Numerous techniques have been developed for the identification of the underlying genetic defect in affected individuals.
Materials and methods: The method consists of: (i) PCR amplification of a single fragment (1087 bp) of the a1 and a2 globin gene flanking all 13 mutations; (ii) three 10-plex primer extension reactions of the unpurified amplification product using allele-specific primers, carrying at their 5’end a unique recognition sequence. The reaction takes place in the presence of biotin-dUTPs and a DNA polymerase that lacks 3’→5’ exonuclease activity and (iii) dry-reagent multi-allele dipstick assay for visual detection of the primer extension reaction products within minutes. The detection of the products is achieved by naked eye using anti-biotin conjugated gold nanoparticles.
Results and conclusions: Parameters that affect the performance of the PCR amplification, PEXT reaction and the multiallele biosensor were investigated in order to optimize the specificity and detectability of genotyping assay. The method was evaluated by analyzing 83 samples of known genotypes and the results were found to be fully concordant with those obtained by the reference methods. The proposed method is simple, rapid, cost-effective, does not require specialized instrumentation or purification of the PCR products and could be a particularly useful tool in laboratories with limited resources.
Descriptive study of genotype frequencies of Ala9Val polymorphism of SOD-Mn in chagasic patients
D'arrigo M (1), Gerrard G (1), Lioi S (1), Zumoffen C (1), Beloscar J (2)
(1) Facultad de Ciencias Bioquimicas Y Farmaceuticas Universidad Nacional de Rosario, Bioquimica Clinica, Rosario, Argentina
(2) Facultad de Ciencias Medicas Universidad Nacional de Rosario, Cardiologia, Rosario, Anguilla
Corresponding author: darrigomabel [at] yahoo [dot] com [dot] ar
Introduction: The existence of individuals infected with Chagas disease (CD) living in endemic areas without apparent heart damage shows that a proportion of them are able to contrarest the infection by Tripanosoma cruzi. Genetic factors of each patient ere actively involved in the evolution of clinical manifestations of CD. In this work we decided to do a descriptive study of genotype frequencies (GF) of Ala9Val polymorphism of SOD-Mn and determine the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in chagasic patients (CP) with cardiomyopathy (CwC) and without cardiomyopathy (CnC) compared with healthy controls (HC).
Materials and methods: The molecular characterization was performed by PCR-RFLP. Enzyme activities were determined by spectrophotometric methods. The hypothesis test under normal theory proportions and Kruskal Wallis test were carried out.
Results: The GF of SOD-Mn (IC 95%) were: HC: Ala/Ala: 0.54, Ala/Val: 0.33, Val/Val: 0.13; CwC: Ala/Ala: 0.35, Ala/Val: 0.30, Val/Val: 0.35; CnC: Ala/Ala: 0.36, Ala/Val: 0.46, Val/Val: 0.18. The enzyme activities were: CAT (K/gHb): HC: 185 ± 28, CwC: 316 ± 68, CnC: 332 ± 41; GPx (U/gHb): HC: 61 ± 1, CwC: 98 ± 17, CnC: 102 ± 20; SOD (USOD/gHb): HC: 895 ± 314, CwC: 3270 ± 833, CnC: 2590 ± 188. The study of SOD-Mn GF and the activities of CAT, SOD and GPx showed significant differences (P<0.01) between CP and HC.
Conclusion: The data reflect that polymorphisms involved in oxidative stress may have implications in the pathogenesis of CD, modifying individual risk in the development of cardiomyopathies.
Endothelial NO-synthase gene polymorphism in pathogenesis of lacunar stroke
Topuzova MP (1), Klocheva EG (1), Zaytseva NO (2), Goldobin VV (1), Vavilova TV (2), Sirotkina OV (2)
(1) North-West State Medical University named after Mechnikov, Neurology, St. Petersburg, Russia (Russian Fed.)
(2) North-West State Medical University named after Mechnikov, Clinical laboratory diagnostics, St. Petersburg, Russia (Russian Fed.)
Corresponding author: marcun [at] rambler [dot] ru
Objective: Determination of endothelial NO-synthase T786S and G894T eNOS polymorphisms in patients with microangiopathy in acute period of ischemic lacunar stroke (LS).
Materials and methods: We examined 67 patients with LS due to microangiopathy (54% female, 46% male, mean age 66 ± 11) and 45 healthy controls. The diagnosis of LS was verified in accordance to current diagnostic criteria, clinically and instrumentally. Genotypes were determined by RT-PCR using the amplifier DT-96 and reagent kit "DNA-Technology" (Moscow, Russia).
Results: The analysis of T786C and G894T eNOS showed the following genotypes distribution: TT in 37.3%, CT in 55.2% and CC in 7.5% patients; GG – 55.2%, GT – 38.8% and TT in 6% patients with LS. Investigation of T786C and G894T eNOS in controls revealed the following genotypes frequencies: TT – 46.7%, CT – 44.4% and CC – 8.9%; GG – 71.1%, GT – 22.2% and TT – 6.7%, respectively. The distribution of T786C eNOS genotype was not significantly different between patients with LS and controls. While, the significant difference was observed in distribution of the G894T eNOS genotypes (P = 0.03). Thereby, the presence of T allele of the G894T eNOS in the homo-or heterozygous state was a risk factor for LS (OR=1.99; CI = 1.07-3.74).
Conclusion: The prevalence of 894T allele eNOS gene in patients with microangiopathy in acute period of ischemic stroke suggests an association of carriage of this allele and development of LS.
Clinical manifestation of hereditary angioedema is less severe in patients with missense mutations
Bors A (1), Varga L (2), Farkas H (2), Tordai A (1), Fust G (2), Szilagyi A (2)
(1) Hungarian National Blood Transfusion Service, Molecular Diagnostics Laboratory, Budapest, Hungary
(2) Semmelweis University, 3rd Department of Internal Medicine, Budapest, Hungary
Corresponding author: bors [dot] andras [at] ovsz [dot] hu
Background: Hereditary angioedema (HAE) is a rare (1:50000), but potentially life-threatening disease, characterized by recurrent, acute edematous episodes, and caused by C1-inhibitor (C1INH) deficiency with an autosomal dominant inheritance. The rate, localization, and severity of symptoms are highly variable among patients. Our aim was to evaluate the genotype-phenotype correlation between disease-causing mutations in C1INH gene and the diverse clinical manifestation of HAE.
Patients and methods: Screening for C1INH mutations was carried out by DNA sequencing, Southern-hybridisation and qPCR based on relative quantification of each exon in 39 unrelated Hungarian HAE type I patients. Patients were followed for a minimum of 5 years and annual frequencies of attacks (subcutaneous, abdominal, upper airway, genital, lingual-labial, erythema) were recorded.
Results: In summary, 7 missense, 4 nonsense, 3 splice site mutations, 1 point mutation in the translation initiation codon, 4 large deletions, 1 small deletion, and 7 frameshift (including 4 previously not reported) mutations were identified. No point mutation or large gene rearrangements were detectable in 6 patients. Annual frequency of attacks, and requirement of C1-inhibitor replacement was significantly lower in carriers of missense mutations (N = 7) compared to patients with non-missense mutations (N = 32, P = 0.008 and P = 0.022, respectively). Carriers of missense mutations had 10 times lower odds ratio for high (> 1/year) attack frequency compared to the rest of patients.
Conclusions: Our findings indicate that missense mutations of the C1INH gene are associated with favourable manifestation of the disease. Information on the mutation type can be used in clinical practice for predicting the severity of HAE.
Serotonin transporter polymorphism (5-HTTLPR) in Croatian population
CulejJ (1), Štefanović M (2), Karlović D (3)
(1) Sestre Milosrdnice University Hospital, Department of transfusiology and haemostasis - Clinic for tumors, Zagreb, Croatia
(2) Sestre Milosrdnice University Hospital, Clinical Institute of Chemistry, Zagreb, Croatia
(3) Sestre Milosrdnice University Hospital, Department of Psychiatry, Zagreb, Croatia
Corresponding author: jelena [dot] culej [at] gmail [dot] com
Background: Serotonin transporter is responsible for serotonin reuptake in the brain as well as other tissues. The most studied polymorphism is consisted of 44 base pairs deletion/insertion in the promoter region (5-HTTLPR) that classifies alleles into „short“ (S) or „long“ (L) variant. Short allele is associated with reduced transcriptional activity. Some previous family based studies support thesis of genetic basis of psychiatric disorders. According to previous research, this polymorphism is related to depression and other psychiatric disorders, as well as with response to selective serotonin reuptake inhibitors treatment. Genetic heterogeneity of population might cause misleading association between cases and controls in case-control studies. The aim of this study was to assess frequency of S and L alleles in Croatian population.
Materials and methods: 307 healthy individuals of both sexes, (age 18 to 50) were included in this study. Their physical and psychical condition was assessed by physical examination, and MINI psychiatric interview for mental disorders exclusion. Genotyping was performed by simple PCR reaction followed by the gel electrophoresis which is used for separation of 375 bp (allele S) from 419bp (allele L).
Results: Genotype frequencies were in accordance with Hardy-Weinberg equilibrium (P = 0.114). L allele frequency is 59% and for S allele 41%. Frequency distribution for studied genotype is: LL – 37%; LS – 44%; SS – 19%.
Conclusion: Based on Hardy – Weinberg equilibrium, investigated population is homogenous and reported genotype frequencies are in accordance to some other studies performed on European population.
Q192R and L55M pon1 gene polymorphisms associated with peripheral arterial disease
KardumParoM (1), PerkovS (1), Flegar- Meštrić Z (1), VidjakV (2), Novačić K (2), Barišić K (3)
(1) Merkur University Hospital, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia
(2) Merkur University Hospital, Institute for Radiology, Zagreb, Croatia
(3) University of Zagreb, Faculty of Pharmacy and Medical Biochemistry, Zagreb, Croatia
Corresponding author: mirjanamariana [at] gmail [dot] com
Background: Development of peripheral arterial disease (PAD) could be due to human serum paraoxonase (pon1) gene polymorphisms or lower catalytic concentrations of serum paraoxonase-1 (PON1). The aim of study was to examine the association of pon1 gene polymorphisms (Q192R and L55M) with the catalytic concentrations of serum PON1 in patients with PAD.
Materials and methods: Healthy subjects (118) and patients with angiographically confirmed diagnosis of PAD (110) were investigated. The catalytic concentration of serum PON1 was determined by spectrophotometric method using paraoxon as the supstrate in the presence of NaCl. pon1 gene polymorphisms were determined by PCR-RFLP method accredited according to ISO15189. The assay performance was assessed by interlaboratory specimens exchange. Allele and genotype frequencies were compared by the χ2 or Fisher exact test.
Results: For pon1 gene polymorphism Q192R allele and genotype frequency were significantly different betweeen patients with PAD and healthy subjects (both < 0.001), while for pon1 gene polymorphism L55M allele (P = 0.649) and genotype
(P = 0.327) frequencies did not differ significantly. The catalytic concentrations of serum PON1 were significantly lower in patients with PAD (P = 0.001). In both groups studied the lowest catalytic concentration were found in QQ and MM genotypes of pon1 gene.
Conclusions: Lower catalytic concentrations of serum PON1 and Q192R pon1 gene polymorphism could play a role in the development of PAD. Differences in the Q and R allelle frequencies between healthy subjects and patients with PAD may be one of the causes for the reduced catalytic concentrations of serum PON1.
Platelet receptor for von Willebrand factor: gene polymorphism and cerebrovascular disease
Laskovets A (1), Zaytseva N (1), Goldobin V (2), Klocheva E (2), Vavilova T (1), Sirotkina O (3)
(1) North-West State Medical University named after Mechnikov, Clinical Laboratory Diagnostics, St-Petersburg, Russia (Russian Fed.)
(2) North-West State Medical University named after Mechnikov, Neurology, St-Petersburg, Russia (Russian Fed.)
(3) B.P. Konstantinov Petersburg Nuclear Physics Institute, Human Molecular Genetics, St-Petersburg, Russia (Russian Fed.)
Corresponding author: laskovetsanas [at] mail [dot] ru
Objective: Platelet activation and aggregation are pivotal in the pathogenesis of cerebrovascular disease. The aim of our study was to determinate the von Willebrand (vWF) factor platelet receptor gene polymorphism in patients with different pathogenetic variants of stroke.
Materials and methods: We examined the T(-5)C and Thr145Met GP Iba in 47 healthy controls and 123 patients with stroke due to macroangiopathy (1 group), microangiopathy (2 group) and subjects with pathological tortuosity of brachiocephalic arteries, but without ischemic stroke (3 group) by PCR-RFLP.
Results: The analysis of T(-5)C GP Iba showed the following genotypes distribution: 75.6% and 24.4%; 65.9% and 34.1%; 82.9% and 17.1%; 78.7% and 21.3% for TT and TC in 1, 2, 3 and control groups, respectively. There was no CC genotype in investigated subjects. The distribution of T(-5)C GP Iba genotype between patients and controls was not significantly different. However, the significant difference was observed in the Thr145Met GP Iba genotypes’ frequencies – 73.2%, 22.0%, 4.8% and 89.4%, 10.6%, 0% for ThrThr, ThrMet and MetMet in 1 group and controls, respectively (P = 0.045). The Thr145Met GP Iba genotypes’ frequencies were not significantly different in 2 and 3 groups – 87.8%, 9.8%, 2.4% and 90.2%, 9.8%, 0%. Thereby, the presence of the 145Met GP Iba allele in the homo-or heterozygous state was a risk factor for stroke due to macroangiopathy (OR = 3.08 [95% CI 1.02-9.33]).
Conclusion: The prevalence of the Thr145Met GP Iba in patients with macroangiopathy suggests an association of carriage of this allele and the development of ischemic stroke.
Li-Fraumeni Syndrome: description of a new pathogenic TP53 mutation
Illana F (1), Llovet P (2), de la Hoya M (2), Olivera H (3), Perez-Segura P (3), Caldes T (2)
(1) Hospital Clinico San Carlos, IdISSC, Laboratory Medicine Department, Madrid, Spain
(2) Hospital Clinico San Carlos, IdISSC, Molecular Oncology Laboratory, Madrid, Spain
(3) Hospital Clinico San Carlos, Department of Medical Oncology, Madrid, Spain
Corresponding author: fran_illana [at] hotmail [dot] com
Background: Li-Fraumeni syndrome is associated to germ-line mutations of one TP53 allele. The most common cancers associated with LFS are breast cancer (30.6%), soft tissue sarcomas (17.8%), brain tumors (14%) and osteosarcome (13.4%). In this work, we found a novel germline TP53 mutation in a high risk breast/ovarian cancer family that's not reported in the P53_IARC database.
Material and methods: We studied BRCA1 and BRCA2 genes in blood of a high risk breast/ovarian family by Denaturing Gradient Gel Electrophoresis and Multiplex Ligation-dependent Probe Amplification. Also, exons 5 to 9 and splice junctions of TP53 were sequenced in blood and tumors of two relative’s patients (30 and 32 years old) who suffered breast cancer (one of them was a Cystosarcoma Phyllodes). Microsatellites were studied in these tumors too.
Results: The whole study of BRCA1 and BRCA2 genes was negative for mutations and deletions/insertions. Sequences of TP53 in germ-line showed a deletion of 9 nucleotides in exon 5 (c.436del9), but it maintains the open reading frame. The deletion is located in binding DNA domain. However, not deletereus effect has been predicted in silico analysis. The study of the TP53 protein by immunohistochemistry (IHC) in the Cystosarcoma Phyllodes showed high expression of the protein. Microsatellites and the sequencing of TP53 in tumors showed the loss of the normal allele (LOH).
Conclusions: The positive IHC, the segregation of the variant with the disease and the LOH of wild type allele in this family indicates that new variant c.436del9; p.Trp146_Asp148del would be pathogenic.
ATP7B gene mutations in Wilson disease patients from Croatia
Ljubić H (1), Božina T (2), Merkler A (1), Relja M (3), Kalauz M (4), Sertić J (1)
(1) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia
(2) School of Medicine University of Zagreb, Department of Medical Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia
(3) University Hospital Centre Zagreb, Department of Neurology, Zagreb, Croatia
(4) University Hospital Centre Zagreb, Department of Internal Medicine, Zagreb, Croatia
Corresponding author: hljubic [at] kbc-zagreb [dot] hr
Background: Wilson disease (WD) is a rare autosomal recessive disorder of copper metabolism associated with progressive liver disease and degenerative changes in the basal ganglia. More than 400 mutations of the ATP7B gene have been proven to cause the absence or dysfunction of copper transporting P-type ATPase. If WD is diagnosed early enough, effective treatments are available that prevent or reverse many manifestations of this disorder.
Materials and methods: 84 unrelated patients, with hepatic, neurological and/or psychiatric symptoms. Biochemical analyses considering copper levels in serum, urine and liver were indicative of WD diagnosis. Genomic DNA was used to amplify 21 exons of the ATP7B gene. Sequencing analysis was performed by PCR and capillary electrophoresis with BigDye Terminator v3.1 kit on Applied Biosystems Genetic analyzer 3130xl. Preliminary screening for most frequent His1069Gln mutation was performed on Roche LightCycler real-time PCR instrument.
Results: Out of total number of WD patients, molecular-genetic analysis confirmed clinical diagnosis in 41 patient (48.8%). 19 (22.6%) patients were found to be heterozygous for certain WD mutation, while in 24 patients (28.6%) no mutations were found. The most frequent mutation in Croatian population is His1069Gln in exon 14 of ATP7B gene, which accounts for 63.3% of total number of WD mutations. Other frequent mutations are mostly located in exons 5, 13, 15 and 21 of ATP7B gene.
Conclusions: Sequencing analysis is the most adequate method for diagnostics of Wilson disease because of its genetic heterogeneity and also in cases with atypical clinical presentation or equivocal copper studies.
Molecular genetic analysis of primary immunodeficiency diseases in Croatian patients
Merkler A (1), Richter D (2), Kelečić J (2), Rako I (1), Marodi L (3), Sertić J (1)
(1) University Hospital Center Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia
(2) University Hospital Center Zagreb, Department of Pediatrics, Zagreb, Croatia
(3) University of Debrecen, Department of Infectious and Pediatric Immunology, Debrecen, Hungary
Corresponding author: amerkler [at] kbc-zagreb [dot] hr
Background: Primary immunodeficiency diseases (PID) are a group of inherited conditions that occur in individuals born with malfunctioned immune system. The aim was to confirm clinical diagnosis at molecular genetic level in patients with PID and to define carrier status by analyzing DNA samples of individuals with PID in family history.
Materials and methods: 15 samples of genomic DNA was analyzed: 7 samples of patients with suspicion on one of the PID and 8 samples of their family members. For identification of mutations in the coding region of analyzed genes, we used the sequencing method on Applied Biosystems 3130xl Genetic analyzer and BigDye® Terminator v3.1 Cycle Sequencing Kit.
Results: In 2 patients with cyclic and severe congenital neutropenia, we found 2 mutations occurred de novo in mother's egg cells. In a patient with suspicion on Omenn syndrom, mutation wasn't found. In 2 patients with X-linked agammaglobulinemia, mutations in the BTK gene were found. In the first patient, mutation occurred de novo in mother's egg cells and, in the other, mutation was inherited from the mother. The third patient with XLA suspicion did not have mutation in the BTK gene. In a patient with suspicion on chronic granulomatous disease, mutation wasn't found, so there is a possibility for further analysis of other genes associated with this disease.
Conclusions: Molecular genetic analysis is the final diagnostic step in confirmation of the PID diagnosis as it provides comprehension of disease mechanisms at the molecular level and correlation between phenotype and genotype of PID.
HLA-DQ genotyping of celiac disease in Mersin Province of Turkey
Polat G (1), Yesiltepe Y (1), Altıntas E (2), Ates F (2)
(1) Mersin University, Faculty of Medicine, Department of Medical Biochemistry, Mersin, Turkey
(2) Mersin University, Faculty of Medicine, Department of Gastroenterology, Mersin, Turkey
Corresponding author: gurbuzp [at] gmail [dot] com
Background: Celiac disease is a common autoimmune disease caused by a chronic inflammatory reaction to the dietary protein gluten in the intestine. It is characterized by gluten-induced symptoms and signs, specific antibodies, specific HLA class II types and enteropathy. The objective of this study is to determine the genetic profile of celiac disease in Mersin Province of Turkey.
Materials and methods: HLA-DQ2, -DQ8 and -DR4 molecular typing was performed in 220 patients. The molecular typing was analyzed using polymerase chain reaction sequence-specific primers and/or reverse polymerase chain reaction sequence-specific oligonucleotides techniques.
Results: We have found HLA-DQ2, -DQ8 and -DR4 types or their combinations in 133 patients. 27.7% of all patients (61/220) were HLA-DQ2, 3.1% (7/220) were -DQ8 and 8.6% (19/220) were -DR4. The combinations were 10.4%, 5.9%, 3.6% and 0.9% for –DQ8 + DR4, DQ2 + DQ8 + DR4, DQ2 + DQ8 and DQ2 + DR4, respectively. 45.8 % of patients showing polymorphisms (61/133) were HLA-DQ2, 5.2% (7/133) were -DQ8 and 14.2% (19/133) were -DR4. The combinations were 17.3%, 9.77%, 6.0% and 1.5% for DQ8 + DR4, DQ2 + DQ8 + DR4, DQ2 + DQ8 and DQ2 + DR4, respectively.
Conclusion: These results support the evidence that HLA-DQ status influences the development of celiac disease. Further, determination of patient’s HLA-DQ genotype allows establishing clinically relevant genetic risk profiles.
Sequencing analysis of CFTR gene in Croatian patients with cystic fibrosis
Zekušić M (1), Ljubić H (1), Juričić L (1), Tješić-Drinković D (2), RakoI (1), Sertić J (1)
(1) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia
(2) University Hospital Centre Zagreb, Department of Pediatrics, Zagreb, Croatia
Corresponding author: mzekusic [at] kbc-zagreb [dot] hr
Background: Cystic fibrosis (CF) is the most common autosomal recessive hereditary disease in Caucasians with an average incidence of 1:2500 newborns. CFTR gene codes for CFTR protein (cystic fibrosis transmembrane conductance regulator) which regulates the transport of chloride ions through cell membranes.
Materials and methods: Genomic DNA of 65 CF patients was screened for 32 mutations by Cystic Fibrosis Genotyping Assay CFv3 (Abbott). Twelve patients who were found to be heterozygous for one CF mutation were further analysed by sequencing method for specific exons (7, 10, 11, 12 and 19) on AB Genetic Analyzer 3130XL, using BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems).
Results: Since year 2001, out of 65 patients detected to have CF, 42 were homozygous with the deltaF508 mutation and 11 were compound heterozygous. By using commercial kit we revealed 14 different mutations in patients with CF. Through sequencing exons 7, 10, 11, 12 and 19 in 12 heterozygous patients so far we have found five polymorphisms: M470V (c.1408A>G), 1898+152T/A (c.1766+152T>A), 3601-65C/A (c.3469-65C>A), 1525-61A/G (c.1393-61A>G) and R75Q (c.224G>A). These DNA polymorphisms will certainly contribute to understanding the sequential variants and clinical correlation. Other CFTR exons still remain to be analyzed.

Conclusions: This paper investigates variants in CFTR gene other than the panel covered by commercial kit. By collecting information about similar cases and studying them, we are likely to notice a link between new mutations, polymorphisms and clinical features.