Attila Tordai

Mutation history of the Roma/Gypsies

The 8-10 million European Roma/Gypsies are a founder population of common origins that has subsequently split into multiple socially divergent and geographically dispersed Gypsy groups. Unlike other founder populations, whose genealogy has been extensively documented, the demographic history of the Gypsies is not fully understood and, given the lack of written records, has to be inferred from current genetic data. In this study, we have used five disease loci harboring private Gypsy mutations to examine some missing historical parameters and current structure. We analyzed the frequency distribution of the five mutations in 832-1,363 unrelated controls, representing 14 Gypsy populations, and the diversification of chromosomal haplotypes in 501 members of affected families. Sharing of mutations and high carrier rates supported a strong founder effect, and the identity of the congenital myasthenia 1267delG mutation in Gypsy and Indian/Pakistani chromosomes provided the best evidence yet of the Indian origins of the Gypsies. However, dramatic differences in mutation frequencies and haplotype divergence and very limited haplotype sharing pointed to strong internal differentiation and characterized the Gypsies as a founder population comprising multiple subisolates. Using disease haplotype coalescence times at the different loci, we estimated that the entire Gypsy population was founded approximately 32-40 generations ago, with secondary and tertiary founder events occurring approximately 16-25 generations ago. The existence of multiple subisolates, with endogamy maintained to the present day, suggests a general approach to complex disorders in which initial gene mapping could be performed in large families from a single Gypsy group, whereas fine mapping would rely on the informed sampling of the divergent subisolates and searching for the shared genomic region that displays the strongest linkage disequilibrium with the disease.

Distinct clinical characteristics of myeloproliferative neoplasms with calreticulin mutations

Somatic insertions/deletions in the calreticulin gene have recently been discovered to be causative alterations in myeloproliferative neoplasms. A combination of qualitative and quantitative allele-specific polymerase chain reaction, fragment-sizing, high resolution melting and Sanger-sequencing was applied for the detection of three driver mutations (in Janus kinase 2, calreticulin and myeloproliferative leukemia virus oncogene genes) in 289 cases of essential thrombocythemia and 99 cases of primary myelofibrosis. In essential thrombocythemia, 154 (53%) Janus kinase 2 V617F, 96 (33%) calreticulin, 9 (3%) myeloproliferative leukemia virus oncogene gene mutation-positive and 30 triple-negative (11%) cases were identified, while in primary myelofibrosis 56 (57%) Janus kinase 2 V617F, 25 (25%) calreticulin, 7 (7%) myeloproliferative leukemia virus oncogene gene mutation-positive and 11 (11%) triple-negative cases were identified. Patients positive for the calreticulin mutation were younger and had higher platelet counts compared to Janus kinase 2 mutation-positive counterparts. Calreticulin mutation-positive patients with essential thrombocythemia showed a lower risk of developing venous thrombosis, but no difference in overall survival. Calreticulin mutation-positive patients with primary myelofibrosis had a better overall survival compared to that of the Janus kinase 2 mutation-positive (P=0.04) or triple-negative cases (P=0.01). Type 2 calreticulin mutation occurred more frequently in essential thrombocythemia than in primary myelofibrosis (P=0.049). In essential thrombocythemia, the calreticulin mutational load was higher than the Janus kinase 2 mutational load (P<0.001), and increased gradually in advanced stages. Calreticulin mutational load influenced blood counts even at the time point of diagnosis in essential thrombocythemia. We confirm that calreticulin mutation is associated with distinct clinical characteristics and explored relationships between mutation type, load and clinical outcome.

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.

Association between Heat Shock Protein 72 Gene Polymorphism and Acute Renal Failure in Premature Neonates

Heat shock protein (HSP)70 plays an important role in the ischemic tolerance of fetal and neonatal kidney. We have investigated the association of genetic polymorphisms of the constitutive HSP70 (HSP73) and the inducible HSP70 (HSP72) encoding genes with the risk of acute renal failure (ARF) in very low birth weight (VLBW) neonates. Thirty-seven VLBW neonates with ARF and 93 VLBW neonates without ARF were enrolled in the study. The presence of HSP72 (1267)AG and HSP73 (190)GC polymorphism was analyzed from dried blood samples by PCR and restriction length fragment polymorphism. Allelic prevalence was related to reference values obtained in 131 healthy adults. Stepwise binary logistic regression was applied to determine the independent effect of the established risk factors to the development of ARF. Sixteen of 37 VLBW neonates with ARF and 18 of 93 VLBW neonates without ARF were homozygous for HSP72 (1267)G allele (p ≤ 0.01). The association between HSP72 (1267)GG genotype and ARF remained at the level of significance (p = 0.05) when it was adjusted for established risk factors of neonatal ARF. Prevalence of HSP72 (1267)GG was also higher in VLBW neonates than in the reference population (p < 0.05) and in VLBW neonates with infant respiratory distress syndrome than in those without (p < 0.001). We found that in VLBW neonates carrying HSP72 (1267)GG genetic variation, which is associated with low inducibility of HSP72, the risk of ARF was increased. Therefore, VLBW neonates with (1267)GG might express less HSP72 and might be less protected against ARF.

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.

The R1141X Loss-of-Function Mutation of the ABCC6 Gene Is a Strong Genetic Risk Factor for Coronary Artery Disease

Loss-of-function mutations of ABCC6 cause pseudoxanthoma elasticum (PXE). This Mendelian disorder is characterized by elastic calcification leading to dermal, ocular, and cardiovascular symptoms like coronary artery disease (CAD) and stroke. Although PXE is a recessive disease, microscopic dermal lesions, serum alterations, and higher anecdotal incidence of stroke or CAD among carriers were reported. Here we investigated the association of the c.3421C>T loss-of-function mutation of ABCC6 and CAD and stroke. A previous study demonstrated the association of the c.3421C>T mutation with CAD; however, the frequency found in the control population was unexpectedly high, contradicting, thus, the prevalence of PXE. In the present study, genomic DNA from 749 healthy blood donors was used as control, while 363 and 361 patients suffering from stroke and CAD were investigated, respectively. One carrier was found in our control group, which is in accordance with the reported prevalence of this mutation. No significant association was found between carrier status and stroke in our cohort. In contrast, a significant association of carrier status and CAD was observed (5/361 carriers: p = 0.016, odds ratio [OR] = 10.5). We propose that carriers of ABCC6 loss-of-function mutations benefit from CAD prevention therapy.

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.

Membrane depolarization selectively inhibits receptor-operated calcium channels in human T (Jurkat) lymphoblasts

Jurkat lymphoblasts were stimulated by a monoclonal antibody against the CD3 membrane antigen and the evoked calcium signal was followed by the intracellular fluorescent calcium indicator indo-1. The technique applied allowed us to separately investigate the stimulus-induced intracellular calcium release and the calcium-influx pathways, respectively. In the same cells membrane potential was estimated by the fluorescent dye diS-C3-(5). The resting membrane potential of Jurkat lymphoblasts under normal conditions was between -55 and -60 mV. Membrane depolarization, obtained by increasing external K+ concentration, removing external Cl-, or by increasing the Na+/K+ leak permeability with gramicidin or PCMBS, did not induce calcium influx in the resting cells and did not influence the CD3 receptor-mediated internal calcium release, while strongly inhibited the receptor-mediated calcium influx pathway. Half-maximum inhibition of this calcium influx was observed at membrane potential values of about -35 to -40 mV and this inhibition did not depend on the external calcium concentration varied between 5 and 2500 microM. Membrane hyperpolarization by valinomycin did not affect either component of the calcium signal. The observed selective inhibition of the receptor-operated calcium influx pathway by membrane depolarization is probably an important modulator of calcium-dependent cell stimulation.

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.