Hajnalka Andrikovics

ATG16L1 and IL23 receptor (IL23R) genes are associated with disease susceptibility in Hungarian CD patients

BACKGROUND North American and European genome-wide association scans have identified ATG16L1 and IL23R as novel inflammatory bowel disease (IBD) susceptibility genes and subsequent reports confirmed these findings in large independent populations. The aims of this study were to investigate the association and examine genotype-phenotype relationships in a Hungarian IBD cohort. METHODS 415 unrelated IBD patients (CD: 266, age: 35.2+/-12.1 years, duration: 8.7+/-7.5 years and UC: 149, age: 44.4+/-15.4 years, duration: 10.7+/-8.9 years) and 149 healthy subjects were investigated. IL23R Arg381Gln (R381Q, rs11209026) and ATG16L1 Thr300Ala (T300A, rs2241880) polymorphisms were tested using LightCycler allele discrimination method. Detailed clinical phenotypes were determined by reviewing the medical charts. RESULTS The association between IL23R rs11209026, ATG16L1 rs2241880 and CD was confirmed (OR(IL23R381Q): 0.38, 95% CI: 0.16-0.87; OR(ATG16L1300AA): 1.86, 95% CI: 1.04-3.40). No difference was found between patients with UC and either controls or CD. In CD, IL23R 381Gln heterozygosity was associated with inflammatory disease (70% vs. 34%, p=0.037), while disease restricted to the colon was more prevalent in patients with the ATG16L1 300Ala/Ala homozygosity (33.3% vs. 21.1%, p=0.036). In addition, carriage of the variant alleles did not predict response to steroids, infliximab or need for surgery. CONCLUSIONS We confirmed that ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Further studies are needed to confirm the reported phenotype-genotype associations found in this study.

High Incidence of Hemochromatosis Gene Mutations in the Myelodysplastic Syndrome: The Budapest Study on 50 Patients

Genotypic testing of nonselected patients with the myelodysplastic syndrome (MDS) for the C282Y and H63D mutations of the HFE gene responsible for hereditary hemochromatosis revealed a significantly increased frequency of these mutations when compared to healthy blood donors reflecting the average population. Among the 50 patients examined [26 refractory anemia (RA), 9 refractory anemia with ring sideroblasts (RARS), 2 refractory anemia with excess of blasts (RAEB) and 13 refractory anemia with excess of blasts in transformation (RAEB-t)] there were 24 heterozygotes (20 for H63D and 4 for C282Y), 1 homozygote for H63D and 1 compound heterozygote. The difference between the HFE-positive and HFE-negative MDS patients as regards initial serum iron and transferrin saturation was not significant. Inevitably the iron overload syndrome eventually develops in MDS patients due to intrinsic characteristics of the disease as well as an escalating need for blood transfusion therapy in the course of the disease. The high incidence rate of HFE gene mutations among MDS patients may also contribute to this vicious circle.

Association of some rare haplotypes and genotype combinations in the MDR1 gene with childhood acute lymphoblastic leukaemia

To investigate their possible roles in disease susceptibility and some disease characteristics we genotyped C3435T and G2677T/A polymorphisms in multidrug resistance-1 (MDR1) gene with a single base extension method and the G34A and C421A polymorphisms of the breast cancer resistance protein gene with an allelic discrimination system in 396 children with acute lymphoblastic leukaemia (ALL) and 192 control patients. While the distribution of individual alleles and genotypes did not differ between patients and controls, there were significant differences in the frequencies of some rare haplotypes and genotype combinations in the MDR1 gene between the two groups.

Synergistic interaction of ABCB1 and ABCG2 polymorphisms predicts the prevalence of toxic encephalopathy during anticancer chemotherapy.

Polymorphisms of the ABCB1 (MDR1) and ABCG2 (BCRP) genes were reported to alter the expression and function of these drug transporters. Both proteins are present at the main pharmacokinetic barriers including the blood–brain barrier. Data from 291 children with acute lymphoblastic leukaemia were analysed in this retrospective study. ABCB1 3435T>C, 2677G>T/A, 1236C>T and ABCG2 421C>A, 34G>A genotypes were determined. Encephalopathy episodes were more frequent among those with ABCB1 3435TT genotype than in the 3435CC/CT group (odds ratio (OR) 3.5; P=0.03). Patients with the ABCG2 421A allele tended to have more complications than wild type homozygotes (OR=2.0; P=0.25). The rate of the adverse effect was similar in those harbouring no or only one of the predisposing genotypes, that is, either ABCB1 3435TT or ABCG2 421AA/AC. However, significantly more children suffered encephalopathy in the group with both predisposing genotypes (OR=12.3; P=0.005). In conclusion, these variations exert synergistic effect in predisposing patients to toxic neurological complications of chemotherapy.

ATP-binding cassette transporter ABCG2 (BCRP) and ABCB1 (MDR1) variants are not associated with disease susceptibility, disease phenotype response to medical therapy or need for surgeryin Hungarian patients with inflammatory bowel diseases

Objective. MDR1 (ABCB1), a member of the ATP-binding cassette (ABC) transporters, is an attractive candidate gene for the pathogenesis of inflammatory bowel diseases (IBD) and perhaps for response to therapy. Since limited data are available on MDR1 and ABCG2 polymorphisms in East European IBD patients, the aim of this study was to investigate ABCG2 and MDR1 variants and responses to medical therapy and/or disease phenotype in Hungarian patients. Material and methods. A total of 414 unrelated IBD patients (Crohns disease (CD): 265, age: 35.2±12.1 years, duration: 8.7±7.6 years and ulcerative colitis (UC): 149, age: 44.4±15.4 years, duration: 10.7±8.9 years) and 149 healthy subjects were investigated. ABCG2 G34A, C421A and MDR1 C3435T, G2677T/A single nucleotide polymorphisms (SNPs) were detected using real-time polymerase chain reaction (PCR). Detailed clinical phenotypes were determined by reviewing the medical charts. Results. The frequency of the ABCG2 and MDR1 SNPs was not significantly different among IBD, CD, UC patients and controls. There was no difference in risk for steroid resistance in CD patients carrying variant ABCG2 (19.6% versus non-carriers 18.4%, p=NS) or MDR1 3435T (CC: 22.2% versus CT/TT: 17.6%) alleles. In addition, carriage of the variant allele was not associated with disease phenotype, presence of extra-intestinal manifestations, smoking, response to infliximab therapy or the need for surgery. In UC, the carriage of variant ABCG2 alleles seemed to be preventive for arthritis (15.5% versus 31.7%, OR: 0.39, 95% CI: 0.16–0.98). Conclusions. MDR1 and ABCG2 SNPs were not associated with disease susceptibility or disease phenotype in Hungarian patients, and variant alleles did not predict the response to medical therapy or the need for surgery. Further studies are needed to clarify the association between the presence of ABCG2 variants and arthritis in UC.

Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.

A case of non-HFE juvenile haemochromatosis presenting with adrenocortical insufficiency [4]

The hepatitis C virus (HCV) has recently been implicated in the pathogenesis of several B-cell lymphoproliferative disorders. In particular, anti-HCV antibodies and/or HCV RNA have been detected in most patients with type II mixed cryoglobulinaemia and in a large proportion of Italian patients with B-cell non-Hodgkins lymphoma (NHL) (reviewed by Silvestri et al, 1996; Agnello, 1997; Ivanovski et al, 1998). Moreover, an asymptomatic monoclonal B-cell expansion has been demonstrated by molecular methods in < 20% of Italian patients with HCV-associated chronic liver disease (Franzin et al, 1995). The high prevalence of chronic HCV infection among patients with B-cell NHL has been observed primarily in Mediterranean countries, particularly in Italy (9±32%), where the prevalence of HCV carriers in the general population is quite high and ranges from 1% to 5 ́4%. A recent US study investigating predominantly Hispanic patients also showed a significantly higher prevalence of HCV infection in NHL patients (22%) than in age-matched controls (Zuckerman et al, 1997). However, other studies from the USA, Canada, Germany, the UK and France have not confirmed this association (reviewed by Germanidis et al, 1999). A possible explanation for this difference is the significantly lower prevalence of HCV infection in the general population of the latter countries. To test this last possibility, we investigated the prevalence of HCV infection in B-cell NHL patients from the Republic of Macedonia, which is characterized by a relatively high prevalence of HCV carriers within the general population (2 ́0%). We tested 112 consecutive patients with NHL (93 Macedonian and 19 Albanian) and a control group of 137 patients with other B-cell malignancies (38 with Hodgkins disease, 43 with chronic lymphocytic leukaemia, nine with acute lymphoblastic leukaemia, 26 with multiple myeloma and one with WaldenstroÈms macroglobulinaemia). The diagnosis was based on histological and immunohistochemical analysis of a lymph node and/or bone marrow biopsy according to the Revised European±American Classification (REAL) of lymphoid neoplasms. The serum samples were tested for antibodies to HCV by third-generation enzymelinked immunosorbent assay (ELISA) using commercially available kits and for HCV RNA by reverse transcriptase± polymerase chain reaction (RT±PCR) amplification of the 5 0 untranslated region of HCV. Table I summarizes the results for the different NHL subtypes and the controls. HCV infection was detected in only one patient with NHL (0 ́89%) and in one of the 137 patients with other B-cell malignancies (0 ́72%). Thus, our study demonstrates a low prevalence of HCV infection in patients with B-cell NHL from Macedonia and a lack of association between the two disorders. The comparable prevalence of HCV infection in the general population of Macedonia with that in Italy indicates that HCV infection requires additional environmental factors and/or a distinct genetic background to induce a malignant B-cell disorder. The same appears to be true for the benign HCV-associated B-cell lymphoproliferations, such as type II mixed cryoglobulinaemia, and the asymptomatic monoclonal B-cell expansions; both disorders were recently found to be significantly less prevalent in Japanese than in Italian patients with HCV-positive chronic liver disease (Pozzato et al, 1999). Therefore, it seems likely that HCV-associated lymphoproliferative disorders have a multifactorial aetiology, which includes additional, presently undetermined, genetic and environmental factors that may vary widely with geography.

The 3′UTR NFKBIA Variant Is Associated with Extensive Colitis in Hungarian IBD Patients

Purpose In previous studies the NFKBIA 3′UTR (untranslated region) AA genotype was associated with Crohn’s disease (CD), while the NFKB1-94ins/delATTG mutation increased the risk for ulcerative colitis (UC). The aim of our study was to investigate these two polymorphisms and patients’ response to medical therapy and/or disease phenotype in Hungarian inflammatory bowel disease (IBD) patients. Methods NFKBIA 3′UTR- and NFKB1-94ins/delATTG polymorphisms were investigated in 415 unrelated IBD patients (CD: 266 patients, mean age 35.2 ± 12.1 years, duration 8.7 ± 7.5 years; UC patients: 149, mean age 44.4 ± 15.4 years, duration 10.7 ± 8.9 years) and 149 controls by PCR-restriction fragment length polymorphism (RFLP) analysis. Detailed clinical phenotypes were determined by reviewing the medical charts. Results The NFKBIA 3′UTR and NFKB1-94ins/delATTG genotypes and allele frequencies were not significantly different among IBD and controls. In patients with UC, the 3′UTR GG genotype was associated with extensive colitis (55.3 vs. 29.4%, odds ratio 2.97, 95% confidence interval 1.45–6.08). The presence of variant alleles did not predict response to steroids, infliximab, or need for surgery. Conclusions The NFKBIA 3′UTR GG genotype was associated with an increased risk for extensive colitis in Hungarian patients. In contrast, variant alleles did not predict response to medical therapy or need for surgery.

Model system for the analysis of cell surface expression of human ABCA1

BackgroundThe ABCA1 protein plays a pivotal role in reverse cholesterol transport, by mediating the generation of HDL particles and removing cellular cholesterol. Both the proper expression of ABCA1 in the plasma membrane and the internalization along with apoA-I are required for function. Therefore, we developed a model system to investigate the effect of clinically relevant drugs on the cell surface appearance of ABCA1.ResultsBy retroviral transduction system, we established stable mammalian cell lines expressing functional and non-functional ABCA1 variants, tagged with an extracellular hemagglutinin epitope. After characterization of the expression, proper localization and function of different ABCA1 variants, we followed quantitatively their cell surface expression by immunofluorescent staining, using flow cytometry. As expected, we found increased cell surface expression of ABCA1 after treatment with a calpain inhibitor, and observed a strong decrease in plasma membrane ABCA1 expression upon treatment with a trans-Golgi transport inhibitor, Brefeldin A. We tested cholesterol level lowering drugs and other potential inhibitors of ABCA1. Here we demonstrate that ezetimibe affects ABCA1 cell surface expression only in the case of a functional ABCA1.ConclusionsOur model system allows a quantitative detection of cell surface expression of ABCA1, screening of substrates or specific inhibitors, and investigating transport regulation.

Thromboembolic Events in Polycythaemia Vera Patients:An Audit of the Hungarian Philadelphia Negative Chronic Myeloproliferative Neoplasia Register

Objective: The Hungarian National Registry for Philadelphia chromosome negative myeloproliferative neoplasms was used to assess the clinical characteristics of Hungarian patients with polycythemia vera. Methods: Data from 351 JAK2 V617F-positive patients diagnosed with PV were collected online from 15 haematology centres reporting clinical characteristics, therapeutic interventions, venous and arterial thromboembolic events, and myelofibrotic or leukaemic transformations. Vascular events (thromboembolic and haemorrhagic) were evaluated before and after diagnosis based upon the Landolfi risk assessment scale. Results: TE were reported on 116 occasions (106 patients) before diagnosis and 152 occasions (102 cases) during follow-up. Compared to before diagnosis, after diagnosis frequency of major arterial events decreased from 11.7% to 2.6% (p<0.0001), and minor venous events increased from 2.0% to 14.2% (p<0.0001); there was no significant change in number of major venous events (from 6.3% to 8.8%; p=0.25) or minor arterial events (from 13.1% to 17.7%; p=0.12). Bleeding events were recorded in 6.4% of patients. Despite treatment, 42.2% of patients with prior thromboembolic events had recurrent thromboembolic complications. After diagnosis age and prior history of thromboembolic events were independent risk factors for arterial events, and white blood cells and diabetes for venous events. Hydroxyurea use in the low+moderate risk Landolfi group slightly, but not significantly, increased thromboembolic event risk (p=0.74). Conclusions: This registry enables characterisation of patients with polycythemia vera. Data suggest the need for accuracy of diagnostic criteria and compliance with risk-adapted therapeutic guidelines.