Zastupljenost mutacija i R202Q polimorfizma gena za porodičnu mediteransku groznicu i njihov uticaj na oksidativni stres i kliničke zapaljenske manifestacije
Distribution of mutations and r202q polymorphism of the gene for familial Mediterranean fever and their influence on oxidative stress and clinical manifestations of inflammation
AuthorRadović, Jelena M.
Committee membersBojanić, Vladmila
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Introduction The Mediterranean fever gene (MEFV) has special importance in autoinflammatory diseases (AID) etiology. The MEFV gene codes for protein pyrin, one of the inflammasome regulators in the innate immune cells. Mutations and R202Q polymorphism of the MEFV gene alter pyrin function, consequently leading to a dysregulation of interleukin-1β release, activation of NF-κB, apoptosis, as well leukocytes oxidative burst. Mutations M680I, M694V, M694I, V726A and E148Q represent the largest percentage of all mutations. Although the R202Q polymorphism in heterozygous form is a benign alteration, several studies confirmed that R202Q homozygotes may develop AID. Familial Mediterranean fever is the most common hereditary autoinflammatory disease with MEFV gene mutations. However, these mutations are determined in a number of other diseases, where they predispose a proinflammatory state, such as: Behcet's disease, Henoch-Schonlein purpura, ulcerative colitis, systemic lupus erythematosus, mu...ltiple sclerosis, etc. The acute phase of AID is caracterised by increased body temperature, abdominal pain, arthralgia, myalgia, erysipeloid rash, fatigue, etc. There is an inflammatory reaction in the blood and increased oxidative stress, detected through the decreased antioxidant activity and increased levels of oxidatively modified molecular products. However, various clinical presentation in the presence of the same MEFV mutations in different individuals, as well as disease in heterozygotes, led to a reconsideration of the role and significance of MEFV gene changes. It is found that mutation carriers have a tendency for febrile episodes, frequent occurrence of rheumatic diseases, and increased levels of acute phase reactants. It is assumed that still unknown modifying genes or environmental factors are partially responsible for AID in heterozygotes. Aim The aim of this study was to investigate the presence and distribution of the MEFV gene mutations and R202Q polymorphism in healthy population in Serbia. Also, the intention was to determine the incidence of recurrent fevers and other clinical signs of inflammation in persons with mutations and R202Q polymorphism, and compare with persons without these changes. In addition, it was investigated whether persons with MEFV changes have altered oxidative stress parameters and what is their association with inflammatory events. Methods The study enrolled 100 healthy subjects. The investigation protocol included: collection of health data (using the official Eurofever project questionnaire) and collection of blood samples to perform genetic test and analysis of oxidative stress parameters. DNA was extracted from peripheral blood leukocytes. DNA sequencing was performed using ABI PRISM 310 automated sequencer (Applied Biosystems, USA). Thiobarbituric acid reactive substances test (TBARS) in plasma was performed by the spectrophotometric method of Andreeva et al. TBARS in erythrocytes were determined by spectrophotometric method of Jain et al. Advanced oxidation protein products (AOPP) were determined spectrophotometricaly and calibrated by chloramine-T solution by the method of Witko-Sarsat V et al. Determination of superoxide dismutase (SOD) was performed with pyrogallol method (McCord J, Fridovich I). Catalase activity was determined by catalase ELISA kit (Enzo Life Sciences), following the manufacturer's instructions. Results Out of 100 examined subjects 11% had MEFV gene mutation: 6% heterozygous E148Q/N mutation and 5% K695R/N. There were 10% of R202Q polymorphism homozygotes and 45% heterozygotes. Unexpectedly, the carrier frequency of mutations and R202Q polymorphism was relatively high for a healthy population in Serbia. Regarding clinical manifestations, persons with the mutations and R202Q homozygotes reported more frequently: febrile episodes of unknown cause (p=0.027), diffuse abdominal pain (p=0.015), peritonitis (p=0.019) and fatigue (p=0.012) in comparison with persons without these gene changes. Individuals with the MEFV gene mutations had more frequently recurrent febrile episodes (p=0.048), diffuse abdominal pain (p=0.017), peritonitis (p=0.008) and fatigue (p=0.032). R202Q homozygotes have experienced significantly more often repeated episodes of fever (p=0.022), diffuse abdominal pain (p=0.009) and lymphadenopathy (p=0.035). Recurrent fever and nonspecific abdominal pain are the most common symptoms in most AID with MEFV gene mutations and suggest the existence of a modified immune response in these persons. Individuals with the MEFV mutations and R202Q homozygotes had significantly higher concentration of erythrocytes TBARS compared with persons without these gene changes (p=0.03), suggesting the existence of increased oxidative damage of erythrocyte membranes. Also, the plasma SOD activity was significantly higher in these individuals (p=0.049), and in R202Q homozygotes particular (p=0.001), which may be due to increased induction of extracellular SOD. Changes in oxidative stress parameters in the MEFV changes carriers were significantly associated with biochemical parameters of inflammation (leukocytes count with erythrocyte TBARS (p=0.009) and erythrocyte sedimentation rate and SOD levels (p=0.031)). Conclusion The MEFV gene mutations are present in 11% of healthy persons in Serbia, those are E148Q/N in 6% and K695R/N in 5% of people. There are 10% homozygote for R202Q polymorphism and 45% heterozygote R202Q carriers in Serbian population. Persons with E148Q and K695R heterozygous MEFV gene mutations and R202Q homozygous polymorphism have frequently febrile episodes of unknown cause, as well as other non-specific manifestations of inflammation. The mutations carriers and R202Q homozygotes have significantly higher values of erythrocyte TBARS and plasma SOD activity, compared with persons without aforementioned genetic changes. Frequent occurrence of non-specific inflammation manifestations in mutation carriers and R202Q homozygotes indicates that these MEFV gene changes are a predisposition for development of inflammation. Changes in oxidative stress parameters indicate the existence of a mild oxidative stress and stimulation of the innate immune response together with possible changes in specific leukocyte functions in these individuals.
Faculty:University of Niš, Faculty of Medicine
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