ALPHA- AND BETA-GLOBIN GENE MUTATIONS IN GEORGIA AND ARMENIA.

Georgia and Armenia are situated at the northern rim of the thalassemia belt and bordering to countries with a known high prevalence of thalassemias. In this study we assessed the carrier frequency and potential spectrum of alpha- and beta-globin mutations among 202 and 190 unselected Georgian and Armenian subjects, respectively. We found four alpha-globin mutations (-3.7del, -4.2del, anti-3.7 triplication, poly-A2) in 9 Armenians (4.74%) and 4 Georgians (1.78%). The heterozygous beta-globin codon 8 [-AA] mutation was detected in one individual from Armenia only. Overall, carrier frequencies seem to be low in both countries, supporting the notion that thalassemias are not a major health problem there.

[Familial Mediterranean Fever in children and adolescents in Georgia].

There are presented preliminary results of the analysis of the materials of the register of children and adolescents suffering from Familial Mediterranean Fever in Georgia. The register was created by the "snow ball" method. For today it contains data on 138 patients, 56 (40.6%) males, 82 (59.4%) females, 86 (62.3%) less than 10 year old, 52 (37.7%) 10-18 year old. Almost in all patients the Armenian roots were revealed, both from maternal and paternal sides. Among the MEFV gene mutations M694V (mainly) and also V726A and M680I were the most common ones.

Guidelines for the genetic diagnosis of hereditary recurrent fevers.

Hereditary recurrent fevers (HRFs) are a group of monogenic autoinflammatory diseases characterised by recurrent bouts of fever and serosal inflammation that are caused by pathogenic variants in genes important for the regulation of innate immunity. Discovery of the molecular defects responsible for these diseases has initiated genetic diagnostics in many countries around the world, including the Middle East, Europe, USA, Japan and Australia. However, diverse testing methods and reporting practices are employed and there is a clear need for consensus guidelines for HRF genetic testing. Draft guidelines were prepared based on current practice deduced from previous HRF external quality assurance schemes and data from the literature. The draft document was disseminated through the European Molecular Genetics Quality Network for broader consultation and amendment. A workshop was held in Bruges (Belgium) on 18 and 19 September 2011 to ratify the draft and obtain a final consensus document. An agreed set of best practice guidelines was proposed for genetic diagnostic testing of HRFs, for reporting the genetic results and for defining their clinical significance.

Mutations in NALP12 cause hereditary periodic fever syndromes.

NALP proteins, also known as NLRPs, belong to the CATERPILLER protein family involved, like Toll-like receptors, in the recognition of microbial molecules and the subsequent activation of inflammatory and immune responses. Current advances in the function of NALPs support the recently proposed model of a disease continuum bridging autoimmune and autoinflammatory disorders. Among these diseases, hereditary periodic fevers (HPFs) are Mendelian disorders associated with sequence variations in very few genes; these variations are mostly missense mutations whose deleterious effect, which is particularly difficult to assess, is often questionable. The growing number of identified sporadic cases of periodic fever syndrome, together with the lack of discriminatory clinical criteria, has greatly hampered the identification of new disease-causing genes, a step that is, however, essential for appropriate management of these disorders. Using a candidate gene approach, we identified nonambiguous mutations in NALP12 (i.e., nonsense and splice site) in two families with periodic fever syndromes. As shown by means of functional studies, these two NALP12 mutations have a deleterious effect on NF-kappaB signaling. Overall, these data identify a group of HPFs defined by molecular defects in NALP12, opening up new ways to manage these disorders. The identification of these first NALP12 mutations in patients with autoinflammatory disorder also clearly demonstrates the crucial role of NALP12 in inflammatory signaling pathways, thereby assigning a precise function to this particular member of an emerging family of proteins whose putative biological properties are currently inferred essentially through in vitro means.

Familial Mediterranean Fever in Armenian population.

Familial Mediterranean Fever (FMF) is an inherited, recessively transmitted inflammatory condition usually occurred in populations from Mediterranean descent (Armenian, Arab, Jewish, Greek, Turkish and Italian populations). Identification of MEFV gene mutations has been of tremendous help for early diagnosis of most cases. The frequency of FMF is different. The prevalence of heterozygous carriers of one of the mutations of MEFV gene is as high as 1 in 5 healthy individuals in Armenia. Genetic testing of this rare Mendelian disorder (MIM no 249100) is efficient for early and prenatal diagnosis of the disease, especially for atypic cases, for carrier screening and pregnancy planning since certain mutations have been shown to have significant correlation with renal amyloidosis (RA), the most severe possible manifestation of FMF. Also genetic testing is very important for colchicine therapy correction. Twelve MEFV mutations are identified in 7000 Armenian FMF patients. Investigation of MEFV mutations in FMF patients (heterozygotes, homozygotes and compound heterozygotes) in comparison with healthy individuals has revealed the most frequent mutations and genotypes, and the information was received about the heterozygous carriers and genotype-phenotype correlation. In heterozygote carriers the most prevalent and severe cases are caused by the presence of a single M694V mutation. Our results could confirm that the MEFV gene analysis provides the first objective diagnostic criterion for FMF (characterisation of the two MEFV mutated alleles in more than 90% of the patients). Molecular testing is also used to screen the MEFV gene for mutations in patients with a clinical suspicion of FMF. We also demonstrated the unfavourable prognostic value of the M694V homozygous genotype, and provided the first molecular evidence for incomplete penetrance and pseudo-dominant transmission of the disease. Overall, these data, which confirm the involvement of the MEFV gene in the development of FMF, should be essential in clinical practice, leading to new ways of managment and treatment of FMF patients.

Molecular study of FMF patients in Armenia.

Familial Mediterranean Fever (FMF, MIM 249100), or Periodic disease, is a recessively transmitted and ethnically restricted condition prevalent in population from the Mediterranean decent. FMF notoriously has been hard to diagnose until mutations in the MEFV gene have been identified and as a tremendous help are used for the diagnosis of difficult cases. Since FMF can be controlled by medication, it is extremely desirable to have a firm diagnosis. The aim of this study was to establish the frequency of the most common mutations and genotypes in Armenian population. Molecular analysis of MEFV gene mutations in 3000 Armenian patients has demonstrated direct correlation between the clinical severity and the molecular diagnostic criteria of the disease, including the development of renal amyloidosis with MEFV genotypes. MEFV genotyping performed in the framework of a genetic counseling may reveal and identify affected individuals in presymptomatic phase, providing the possibility of a precocious start of the therapy.

Oxyradical-mediated chromosome damage in patients with familial Mediterranean fever.

Increased chromosome breakage is observed in patients with familial mediterranean fever (FMF). Their plasma contains clastogenic material inducing chromosome damage in cells from healthy persons. It is proposed that increased oxyradical generation by activated polymorphonuclear cells in blood and serosal fluids of these patients leads to the formation of a clastogenic factor (CF), as it is observed in other chronic inflammatory diseases. Also similar to these diseases, the clastogenic effects are prevented by superoxide dismutase and partially by inhibitors of arachidonic acid metabolism.

Radiation-induced clastogenic factors: anticlastogenic effect of Ginkgo biloba extract.

Clastogenic factors (CFs) were first described in the blood of persons irradiated accidentally or for therapeutic reasons. Work of our laboratory has shown that they occur also under other circumstances, which are characterized by oxidative stress, and that CF-induced chromosome damage is regularly prevented by superoxide dismutase (SOD). Recently we found CFs in a high percentage of salvage personnel of the Chernobyl reactor accident. These liquidators represent a high-risk population and might benefit from cancer chemoprevention by antioxidants. SOD would have to be injected and is not appropriate for long-term prophylactic treatment. In the present study, we therefore evaluated the anticlastogenic effect of the Ginkgo biloba extract EGb 761, which is known for its superoxide scavenging properties. EGb 761 was tested on CF-treated blood cultures of healthy donors. After establishing the optimal protective EGb concentration, using CFs produced by irradiation of whole blood from healthy volunteers, the extract was tested on cultures exposed to CFs from plasma of persons irradiated as liquidators. The anticlastogenic effect could be confirmed for a final concentration of 100 micrograms/ml. In 12 consecutive experiments, CFs induced an average of 18.00 +/- 4.41 aberrations/100 cells. This was reduced to 7.33 +/- 3.08 in the parallel cultures receiving 100 micrograms/ml EGb 761 (p < .001). SOD was anticlastogenic in the same system at concentrations of 30 cytochrome C units/ml (approximately 10 micrograms/ml). Preliminary results obtained in a small series of liquidators showed regression or complete disappearance of CFs in the plasma after 2 months of treatment with EGb 761 (3 x 40 mg/d).

Transferable clastogenic activity in plasma from persons exposed as salvage personnel of the Chernobyl reactor.

Clastogenic factors were first described in the plasma of people who had been accidentally or therapeutically irradiated. They were found also in A-bomb survivors, where they persisted for many years after the irradiation. The present study searched for these factors in the plasma of 32 civil workers from Armenia, who had been engaged as "liquidators" around the Chernobyl atomic power station in 1986. It also included 15 liquidators who had emigrated from the ex-Soviet Union to Israel. Reference plasma samples were obtained from 41 blood donors from the Armenian Blood Center in Yerevan. The samples were tested for their clastogenic activity in blood cultures from healthy donors. The majority of results from the liquidators exceeded those from the unexposed reference samples. The samples from the first Armenian group, with the higher average irradiation dose (0.6 +/- 0.6 Gy), were more clastogenic than those from the second group exposed to 0.2 +/- 0.2 Gy. The number of aberrations in the test cultures was 17.9 +/- 2.9% and 10.5 +/- 3.8% respectively, compared to 5.7 +/- 3.2% in the cultures exposed to the reference ultrafiltrates from Armenian blood donors. The samples from the Israeli liquidators also induced significantly increased aberration rates (14.0 +/- 3.9% aberrant cells). The clastogenic activity was regularly inhibited by superoxide dismutase, indicating that the chromosome-damaging effects of radiation-induced clastogenic factors are exerted via the intermediation of superoxide radicals, as is known for clastogenic factors of different origin.

Clastogenic factors in the plasma of Chernobyl accident recovery workers: anticlastogenic effect of Ginkgo biloba extract.

Clastogenic factors are found in the plasma of persons irradiated accidentally or therapeutically. They persisted in the plasma of A-bomb survivors over 30 years. Clastogenic factors were found in 33 of 47 Chernobyl accident recovery workers (often referred to as liquidators) in a previous study (I. Emerit et al., J. Cancer Res. Clin. Oncol. 120, 558-561, 1994). In the present study, we show that there is a positive correlation between clastogenic activity and dose and that these biomarkers of oxidative stress can be influenced successfully by appropriate antioxidant treatment. With the authorization of the Armenian Ministry of Health, 30 workers were treated with antioxidants from Ginkgo biloba leaves. The extract EGb 761 containing flavonoids and terpenoids was given at a daily dose of 3 x 40 mg (Tanakan, IPSEN, France) during 2 months. The clastogenic activity of the plasma was reduced to control levels on the first day after the end of the treatment. A 1-year follow-up showed that the benefit of the treatment persisted for at least 7 months. One-third of the workers again had clastogenic factors after 1 year, demonstrating that the process which produced clastogenic factors continued. However, the observation that antioxidants do not have to be given continuously is encouraging for intervention trials on a large-scale basis. These appear justified, since clastogenic factors are thought to be risk factors for the development of late effects of irradiation.

[Modelling of a modification of chemical mutagenesis in human cells. II. The dependence of the effect on the time of culture treatment]. / Modelirovanie modifikatsii khimicheskogo mutageneza v kletkakh cheloveka. Soobshchenie II. Zavisimost' éffekta ot vremeni obrabotki kul'tur.

The influence of 2,3-aminopropylaminoethylthiophosphoric acid (2,3-APAETP) on the effect of the alkylating agent, thio TEPA, is investigated at different times of cultivation of human peripheral lymphocyte culture. The analysis of correlation equations shows that the results are described by the polynomes of 4-degree in variants treated with thioTEPA and 2,3-APAETP. The protector effect of 2,3-APAETP is determined not by the time from the culture stimulation but the time between the moment of culture treatment with 2,3-APAETP and thioTEPA and the moment of the fixation. It is shown that the points of maximal sensitivity of the cell cycle to thioTEPA and to the protective effect of 2,3-APAETP are similar.

[Modeling modification of chemical mutagenesis in human cells. 1. Elimination of chromosomal aberrations]. / Modelirovanie modifikatsii khimicheskogo mutageneza v kletkakh cheloveka. Soobshchenie I. Eliminatsiia aberratsii khromosom.

Elimination of chromosome aberrations was studied in populations of dividing cells. For this purpose, on the basis of the corresponding theory of Carrano-Heddle assuming the Poisson distribution, a theory is advanced by the authors based on geometrical distribution, describing the distribution of lesions caused by the action of tioTEF. Parameters of elimination are obtained observed in case of addition of a protector, aminopropy-laminoethylthiophosphoric acid (APAETF) in a lymphocyte culture of human peripheral blood. It is shown that the addition of the protector diminishes the probability of the transmission of chromosome aberrations to daughter cells.

[Effect of interferon on the number of cytogenetic disorders occurring in human cultured lymphocytes treated with thio-TEPA and fotrin]. / Vliianie interferona na kolichestvo tsitogeneticheskikh narushenii, voznikaiushchikh v kul'tiviruemykh limfotsitakh cheloveka pri obabotke tioTEF i fotrinom.

The paper deals with the modifying effects of natural (leukocytic) and synthesized (recombinant) interferons on the number of cytogenetic injuries in the cultured lymphocytes of human peripheric blood after exposure to alkylating chemicals--thio-TEPA and fotrin. The analysis of chromosomal aberration levels is suggestive of a significant protective effect exerted by interferons. The addition of the recombinant interferon increased the number of sister chromatid exchanged frequency in mutagen treated variants. The application of the test system that involves two types of interferon made it possible to reveal differences in their cytogenetic effect during the protector-sensitive period of cultivation.

[Intergroup differences in anticlastogenesis]. / Mezhgruppovye razlichiia antiklastogeneza.

The paper deals with the mechanisms of anticlastogenesis protection of chromosomes in cultured human cells, which depend on many external and internal factors and may vary in genetic risk groups and in individuals. It also analyzes the role of repair and metabolic disturbances in the observed inter- and intragroup differences in the efficiency of protective action in model test systems. Results of comparative analysis of the efficiency of various interferons in the cultured cells from patients with bronchial asthma are presented. The paper first provides evidence for attenuated protective effects in the cultured lymphocytes from patients with xeroderma pigmentosum or chronic recurrent urticaria as compared to those from healthy donors. Prospects of and optimal methodological approaches to the study of anticlastogenesis in human cells are discussed.

[Cytogenetic damages in the cells of allergy patients]. / Izuchenie tsitogeneticheskikh povrezhdenii v kletkakh bol'nykh allergiei.

The levels of cytogenetic damages in cell cultures of chemical industry workers suffering from different forms of allergy have been investigated. The levels of chromosomal aberrations in cells of allergic patients are shown to reliably increase as against those of healthy donors. Studies on SCE levels have shown no such differences between the patients and donors. The SCE levels have been determined at different periods of BUdR addition and cultures fixation under the effect of environmental factors.

[The cytogenetic effect of natural modifiers of mutagenesis in a human lymphocyte culture: the modification of the mutagenic effect by recombinant interferon in vitro in the lymphocytes of bronchial asthma patients and of healthy donors]. / Tsitogeneticheskii éffekt estestvennykh modifikatorov mutageneza v kul'ture limfotsitov cheloveka: modifikatsiia mutagennogo éffekta rekombinantnym interferonom in vitro v limfotsitakh bol'nykh bronkhial'noi astmoi i zdorovykh donorov.

The significant protective effect of recombinant interferon in the cultures of lymphocytes of healthy donors and patients with bronchial asthma has been revealed. The cytogenetic damage were stimulated by alkylating agents thioTEPA and photrin during their administration at the stages Gi-S of the cell cycle. No differences were revealed in the action of mutagens and protector in the patients and healthy persons.

PYPAF1 nonsense mutation in a patient with an unusual autoinflammatory syndrome: role of PYPAF1 in inflammation.

OBJECTIVE: To gain insight into the pathophysiology of an unusual autoinflammatory syndrome, in a patient of Armenian origin, that mimicked familial Mediterranean fever (FMF) but with episodes triggered by generalized exposure to cold, and to further elucidate the controversial function of the protein encoded by PYPAF1, whose mutations (exclusively missense to date) have been identified in 3 hereditary recurrent fever syndromes. METHODS: The patient's DNA was screened for mutations in both MEFV, the gene responsible for FMF, and PYPAF1. The ability of different recombinant PYPAF1 isoforms, expressed in HEK 293 cells, to regulate NF-kappaB signaling was subsequently assessed. RESULTS: No disease-causing mutation was found in MEFV. However, a nonsense mutation (p.Arg554X) was identified in PYPAF1; this defect resulted in a truncated protein lacking all leucine-rich repeats. Study of the wild-type and mutant PYPAF1 recombinant proteins revealed that PYPAF1 inhibited NF-kappaB proinflammatory pathways, and that the identified nonsense mutation impaired this property. CONCLUSION: These molecular and clinical findings, together with the clinical manifestations in the patient, which call into question the current nosology of the hereditary recurrent fever syndromes, are consistent with the hypothesis that PYPAF1 acts as an inhibitor of NF-kappaB signaling. They also provide a clear elucidation of the functional consequences of this nonsense PYPAF1 mutation not previously described in the literature, which result in a partial loss of function and may thereby explain the pathophysiology of the autoinflammatory syndrome observed in this patient.

Familial Mediterranean fever: clastogenic plasma factors correlated with increased O2(-)--production by neutrophils.

Familial Mediterranean fever (FMF) is an autosomal recessive disease predominantly affecting Armenians and non-Ashkenazi Jews. The disease begins in childhood with paroxysmal attacks of pain and fever accompanied by peritonitis, pleuritis, and synovitis. During the acute phase, there is a massive influx of polymorphonuclear leukocytes into the serosal membranes, connected with degranulation of the neutrophils and with secretion of lysosomal enzymes and pyrogenic substances. An increase in the lipoxygenase product, leukotriene B4, a chemotactic agent, and a decrease in the activity of the inhibitor of chemotaxis, C5a, in serosal fluids have been considered responsible. Previous work from our laboratories had shown that the chromosomal instability observed in blood cultures of patients with FMF is secondary to circulating clastogenic factors (CFs), and that the antioxidant enzyme superoxide dismutase, as well as lipoxygenase inhibitors, reduce the chromosome damaging effects. CFs are observed in chronic inflammatory diseases and in various other pathological conditions accompanied by oxidative stress. Similar clastogenic materials were found in supernatants of neutrophils and monocytes after a respiratory burst and were shown to contain lipid peroxidation products and cytokines. In the present study we compared the clastogenic effects exerted by plasma ultrafiltrates from 20 adult patients with FMF to the unstimulated O2- production of their neutrophils. In comparison to 20 age- and sex-matched controls, which were studied simultaneously, the O2- production by patient's neutrophils was routinely higher than that of controls. The clastogenic effects of patient's plasma, expressed as the number of chromosomal aberrations induced in test cultures of healthy donors, were correlated with the importance of O2- production by their neutrophils (r = 0.5235). Even if the relative contribution of disturbance in arachidonic acid metabolism, neutrophil activation, and CF formation in the disease process remains unclear, the demonstration of oxidative stress in this genetic disorder suggests the use of antioxidants and free radical scavengers, in particular during acute attacks, when the classical colchicine treatment is without effect.