RISK FACTORS OF AMYOTROPHIC LATERAL SCLEROSIS IN GEORGIA.

Objective - to identify risk factors in patients diagnosed with Amyotrophic Lateral Sclerosis in Georgia directed to The First University Clinic of TSMU and P. Sarajishvili Institute of Neurology. Totally 53 patients, aged 24 to 82 years, were investigated with Amyotrophic Lateral Sclerosis (ALS), defined by "Gold Coast " criteria. We have used the Questionnaire for Environmental Exposures, Toxins, and Neurological diseases developed by Dartmouth-Hitchcock Medical center to identify risk factors, and categorized patients according to the place of settlement and environmental hazards. The control consisted of age and sex matched 50 healthy individuals. The brain was visualized by MRI (1.5T), and Electromyography (EMG) was performed on all patients. ALS risk was higher among those ever holding a job in mechanics, painting, or construction (p<0.05), head trauma or concussion that caused a "blackout" or loss of consciousness was associated with a higher risk of ALS (p<0.01). Demographically more ALS cases were found in Tbilisi and Imereti, compared to other regions (p<0.05). According to our research on Georgian ALS cases, several occupational jobs, Head trauma is associated with developing ALS in Georgia, Research is needed to identify environmental risk factors attributing to higher rates of ALS in Tbilisi and Imereti.

CLINICAL CHARACTERISTICS OF ALS IN GEORGIAN PATIENTS.

Amyotrophic Lateral sclerosis (ALS ) is a fatal progressive neurodegenerative disease that affects the upper and lower motoneurons. .The disease is characterized by a plethora of neurological symptoms. There is a lot of information in the medical literature about ALS phenotypes, but the clinical diversity of ALS has not been studied in the Caucasus region and a unified clinical picture has not been conclusively established. In this regard, it is very important to study the symptoms among patients with ALS in Georgia. From 2018 to 2021, we examined 47 patients with ALS living in Georgia from different parts of the country, 23 - female, 24 - male, diagnosed based on clinical picture, electromyographic studies (AWAJI) and who met the EL ESCORIAL -Revised criteria. Also clinical symptom studies were conducted using the Mayo Clinic Laboratory Neurological Questionnaire. Cognitive changes were assessed using Addenbrooke's Cognitive Examination scale (ACE III) and the Frontal Behavioral Questionnaire, the patient's quality of life was assessed by ALSFRS-R. Patients were 26 to 84 years old, the age of onset of the disease was 58-60 years in men, 55-57 years in women. The bulbar type was observed in 21.3%, the upper limb type in 38.3% and the lower limb type in 40.4%. Frontotemporal dementia (FTD), diagnosed in 6 patients (12.7%). No reliable correlation was found between the forms of ALS and FTD. The results of the study showed that ALS is a multisystem disease and is not limited to damage to motoneurons. It is safe to say that ALS has characteristics of polysystemic degeneration, with the predominance of motorneuron damage. Therefore, we consider it advisable to screen all patients with ALS for additional symptoms with a focus on the examination of cognitive function, which ensures the proper management of the disease in the future.

ASSESSMENT OF THE VALUE OF METHYLATION FEATURES IN DIFFERENT TISSUES FOR PREOPERATIVE IDENTIFICATION OF HIGH-RISK BREAST TUMORS.

Epigenetic changes in breast cancer patients have long been believed to be a promising marker for clinical management - early detection, prognostication, etc. Various approaches are available to test global DNA or target gene methylation status. We used some of them in different tissues of patients with breast cancer to analyze possible links between epigenetic parameters and their value for predicting clinicopathological characteristics (most importantly stage and lymph node status) of tumors. Patients with ductal and lobular invasive carcinoma were included in the study along with age-matched controls. Blood, tumor tissue and normal breast ductal epithelial cells were investigated using MS-HRM technique (for BRCA1 gene promoter methylation assessment), COBRA-PCR (for Alu element methylation quantification) and ELISA-based method (for global DNA methylation estimation). These parameters were analyzed in comparison to clinical, histologic and phenotypic characteristics of the tumors. BRCA1 promoter methylation was detected exclusively in tumor tissues and in only two cases, both of which had aggressive phenotype. Alu and global DNA methylation showed markedly low levels in all tissues of cancer patients compared to healthy controls, with extreme hypomethylation in tumor tissue. Changes in these two parameters were not always concordant. Methylation levels detected in blood showed rather weak relationship with clinicopathological characteristics of tumors. We could conclude that tested parameters are not useful for preoperative determination of tumor stage or lymph node status, neither any other clinicopathological characteristics of tumors. Epigenetics signatures of different tissues of the same patient are not homogenous. Given the uniform picture of DNA methylation in blood in some of investigated groups, additional data and very careful approach are required when considering using it as a diagnostic or predictive tool.

LINE-1 METHYLATION IN BLOOD AND TISSUES OF PATIENTS WITH BREAST CANCER.

Methylation is an epigenetic alteration proved to be involved in many disease processes including cancer. This change affects mainly gene promoters and repetitive sequences in genome. Long Interspersed Nuclear Element-1 (LINE-1) is a family of retrotransposons - repetitive elements that modify gene activity and can themselves be targeted by epigenetic mechanisms. LINE-1 methylation level is a surrogate marker for global methylation. In many conditions this parameter is found to be altered not only in affected cell groups, but also throughout other tissues. The aim of our study was to compare LINE-1 methylation pattern in DNA extracted from blood of the patients with benign and malignant breast tissue. In addition, we investigated correlation of LINE-1 methylation in blood and tissues of same patients and relationship of all variables with histopathologic and phenotypic characteristics of tumors. Patients with biopsy-proved ductal invasive carcinoma of breast and no preoperative chemo/radiotherapy were chosen for the study group. Another pool of patients with various benign breast lesions represented controls. Blood samples from both group members were collected preoperatively. Tumor tissue sections were processed for pathology report and part of remaining tissue was used for methylation study. LINE-1 methylation level was quantified using ELISA-based assay. It was analyzed in combination with histologic and phenotypic tumor parameters and compared between different tissues and different study groups. LINE-1 was found to be significantly hypomethylated in breast cancer tissue compared to blood. Blood samples of patients with malignant tumors showed slightly lower methylation level, than samples obtained from control group members. Lymphovascular invasion was the only aggressiveness-determining factor that was found to be at least weakly correlated with LINE-1 hypomethylation in blood. We can conclude, that global hypomethylation measured by LINE-1 methylation level is significant in tumor tissue. But there is no significant difference between LINE-1 methylation levels in blood of patients with benign and malignant breast tumors; therefor LINE-1 hypomethylation in blood cannot be used as a marker for early tumor detection. Neither is it valid for determination of tumor behavior.

ALTERATION IN THE CYTOKINE SECRETION PATTERN IN T CELLS OF PATIENTS WITH CYSTIC FIBROSIS CAUSED BY DNA METHYLTRANSFERASE INHIBITOR 5-AZACITIDINE.

Cystic fibrosis (CF) is the autosomal-recessive disorder caused by mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The Airway inflammation plays a central role in the progression of CF disease. Cystic fibrosis characterized by the overproduction of the pro-inflammatory cytokines and reduced expression of anti-inflammatory cytokines. Although the mechanisms of abnormal cytokine expression is still poorly understood, altered epigenetic regulations in T cells might contribute. In the present study we examined the expression of IFN-γ and IL-10 by CF T cells prior to and following 5-azaC treatment. In addition we investigated DNMTs levels in nuclear extracts of CD4+ T cells derived from CF and non-CF individuals. Seven CF patients (age: 5-12 years) were included in the study and compared to six age-matched healthy subjects (age: 6- 13 years). CD4+ T cells were isolated from PBMC using CD4 MicroBead kit (Miltenyi Biotec GmbH) and were cultured in RPMI 1640 medium at 37°C with 5% CO2, in presence or absence of 5-azacytidine. Concentrations of IL-10 and γ-INF in CD4+ T Cells were measured by ELISA (eBoiscience, san Diego, CA, USA). In our study we showed that 5 Azacytidine alters nuclear levels of DNMT 3a as well as modulates cytokine levels in CD4+ T cells derived from CF patients. After 5-azaC treatment secretion of IFN-γ was significantly decreased in CF T cells, while amount of IL-10 was elevated by ~2.5 times compared to untreated controls (P<0.05). In summary, data presented in this report demonstrates that epigenetic mechanisms such as DNA methylation may be considered as a one of the potential therapeutic target in a treatment of Cystic Fibrosis.

MTHFR GENE C677T POLYMORPHISM AND LEVELS OF DNA METHYLTRASFERASES IN SUBCLINICAL HYPOTHYROIDISM.

The aim of our study was to investigate the link between MTHFR gene C677T polymorphism and DNMTs levels in patients with Subclinical Hypothyroidism (SCH). In this study 19 adult patients with subclinical hypothyroidism and 19 healthy controls (mean age 31±5.5 and 33±5.1 years respectively) were recruited. All patients were diagnosed based on serum levels of TSH, FT4, anti-TG and anti-TPO antibodies. Written informed consents were obtained from all study subjects. Genomic DNA was extracted using Quick-DNA Universal Kit (Zymo Research, USA). The MTHFR C677T polymorphism was genotyped by PCR-RFLP method. Levels of DNMT1 and 3a were measured in nuclear extracts of PBMC using DNMTs assay kits (Abcam). Our data indicates that the frequency of genotypes and alleles were different among the patient and the control group. There is a significant increase in CC genotype distribution in the control group when compared to the SCH patient group, while the CT as well as TT genotype distribution were not increased significantly in SCH group versus control group. However the C allele is significantly prevalent in the control group compared to the SCH group, while T allele is prevalent in patients compared to the control group with a statically significant difference. In addition, individuals with TT and CT genotypes and hypothyroidism showed elevated amount of DNMT3a in nuclear extracts of PBMC compared with controls, while no significant difference in DNMT1 levels was observed. This study indicates the MTHFR C677T variant may contribute in alteration of epigenetic regulation such as DNA methylation mediated by DNA methyltransferases in patients with subclinical hypothyroidism and also, carriers of the T allele might have an increasing risk of developing SCH.

A STUDY OF THE RELATIONSHIP BETWEEN LEVELS OF METHYLTRANSFERASES IN PERIPHERAL BLOOD MONONUCLEAR CELLS AND CHARACTERISTICS OF TUMOR IN PATIENTS WITH DUCTAL INVASIVE CARCINOMA OF BREAST.

The role of epigenetics in tumor development and progression is actively being studied. The aim of our current pilot study is to analyze correlation of changes in the levels of methyltransferases in nuclear extracts of blood cells with some morphological and phenotypic characteristics of breast cancer. The levels of DNMT1, DNMT3a and H3K4 methyltransferase were measured. The results showed that the level of DNMT1 was highest in the control group but correlation with the tumor grade was just moderate. DNMT3a was found in highest level in Grade III cancer group, followed by Grade II and Grade I groups. Correlation of DNMT1 level with tumor grade was moderate. An opposite pattern was seen for H3K4 methyltransferase. DNMT3a level was higher in larger tumors, while the level of H3K4 methyltransferase was lowest in large tumors with significant negative correlation with the tumor size. This primary study shows that there are some changes in methyltransferase levels in PBMC from breast cancer patients. These changes are most probably attributed to modification of initiation as well as sustainment of methylated status of products.

Epigenetic regulation of acute inflammatory pain.

Acute pain is associated with tissue damage, which results in the release of inflammatory mediators. Recent studies point to the involvement of epigenetic mechanisms (DNA methylation) in the development of pain. We have found that during acute inflammatory pain induced by the application of 10% mustard oil on the tongues of rats, levels of DNMT3a and 3b were elevated markedly (36 and 42 % respectively), whereas the level of DNMT1 was not changed significantly. Previous injection of Xefocam with 0,4 mg/kg dose decreased levels of DNMT3a and 3b (25 and 24% respectively). The level of DNMT1 was not changed significantly compared to the control group. The findings support the idea that inhibitors of DNA-methyltransferases could be useful for pain management. Our data suggest that NSAIDs (alone or in combination with DNMT inhibitors) may be proposed as possible epigenetic regulatory agents, which may play a role in epigenetic mechanisms indirectly through altering the activity of inflammatory mediators involved in pain development.

Expression pattern of DNA-methyltransferases and its health implication (short review).

Epigenetics is heritable and reversible alterations of gene expression without direct alteration of DNA sequences. One example of epigenetic factors is DNA methylation, which prevents certain genes from being expressed. Another example is histone modifications. In addition, miRNAs can silence genes at transcriptional and posttranscriptional level. DNA methylation is regulated by DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b). Aberrant DNMTs expression is the dominant mechanism for the genome instability which associates with a wide range of diseases such as a cancer, autoimmune diseases, mental disorders. In this article we reviewed the major mechanisms of changes of DNA methylation regulated by DNMTs and the role of this changes in pathogenesis of various diseases. In addition we briefly reviewed epigenetic agents, such as inhibitors of DNA methyltransferases or HDAC (histone deacetylase) targeting oncology, hematology, immunology, and neurologic disease indications, and which are in various phases of study or have been clinically tested and approved by FDA (Food and Drug Administration).

mGluR1 interacts with cystic fibrosis transmembrane conductance regulator and modulates the secretion of IL-10 in cystic fibrosis peripheral lymphocytes.

Cystic fibrosis (CF) is caused by the mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. CFTR dysfunction in T cells could lead directly to aberrant immune responses. The action of glutamate on the secretion of IL-8 and IL-10 by lymphocytes derived from healthy subjects and cystic CF patients, as well as the expression of metabotropic glutamate receptor subtype 1 (mGluR1) in the membrane fractions of lymphocytes was investigated. Our results have shown that CF-derived T-cells in the presence of IL-2 produce more IL-8 and IL-10, than T-cell from healthy control. However, only in normal lymphocytes a significant increase (144%) in the IL-10 secretion during exposure to high concentration of glutamate (10(-4)M) was detected. Glutamate-dependent secretion of IL-10 was not inhibited either by NMDA-receptor (NMDAR), or by AMPA-receptor (AMPAR) antagonist. Only mGluR1 antagonist, LY367385, strongly decreases the production of IL-10. Furthermore, the content of mGluR1, as well as cystic fibrosis transmembrane conductance regulator-associated ligand (CAL), Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), was analyzed in plasma membrane of lymphocytes after immunoprecipitation of CFTR. We have found that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with metabotropic mGluR1, but the level of surface exposed mGluR1 in CF-lymphocytes was much lower than in normal cells. Besides, our results have shown that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with NHERF-1 and CAL; however in lymphocytes with CFTR mutation the amount of cell-surface expressed CFTR-CAL complex was greatly decreased. We have concluded that CFTR and mGluR1 could compete for binding to CAL, which in turn downregulates the post-synthetic trafficking of mGluR1 and decreases the synthesis of IL-10.

Rare tubulopathy - primary hypophosphatemic rickets (case report).

UNLABELLED: Primary hypophosphatemic rickets is a rare disorder caused by inborn defect of renal tubular reabsorbtion and usually manifested in childhood and infancy with stunted growth and deformities of lower limbs. Patient 12 years old, female, was born to healthy parents, at 41 weeks of gestation by normal delivery and had a normal birth weight and length. She had one healthy sibling (male). Psychomotor development until the age of 2 years was normal. Since the age of 2 years deformation of legs and difficulties with walking have been observed. The Patient was consulted by Pediatric Orthopedist, Nephrologists and Endocrinologist. The blood biochemical findings revealed normal pH, normal calcium (CA), potassium (K), sodium (Na) concentrations, very low phosphate (P) with markedly elevated alkaline phosphatase (AP) and slightly elevated parathyroid hormone (PTH) concentration; urine test indicated impaired tubular function: mild glucosuria, proteinuria, and markedly increased phosphaturia. Phosphate tubular reabsorbtion (PTR) was 52%, phosphate/creatinine clearance 0,52, renal threshold phosphate concentration (TmP/GFR) 0,25 mmol/l. Genetic tests results not available yet. RESULTS: the diagnosis of phosphate diabetes made on the basis of clinical-laboratory data. The therapy with Inorganic phosphate (50-100mg/kg/d) and 1,25(OH)D3 40-50 ng/kg/d had been started. Childs walking abilities improved with treatment, the deformation of legs decreased, but O-legs and other skeletal deformations are still remarkable. Auxological parameters are not satisfactory. Physical growth is below the normal range (height SDS -3,78); the patient's predicted height less than target height. Biochemical monitoring performed regularly under the treatment, serum phosphate remains below normal, serum ALP is still elevated. CONCLUSION: This case is in line with other publications and indicates the difficulty to achieve normal phosphate levels and normal growth without an additional treatment with growth hormone. At this stage of the disease, the question of referring to the growth hormone therapy is being considered aimed to improve the growth tendencies. It is possible that orthopedic surgery is also needed in the future.

Glutamate decreases the secretion of IL-10 by peripheral blood lymphocytes in persons with autoimmune thyroiditis.

Human T lymphocytes expose ionotropic and metabotropic glutamate receptors, which control immune responses, cell activation, maturation, and death. Several cytokines release during inflammation which identification may have important physiological and clinical implications. Main biological function of IL-10 is limitation and termination of inflammatory responses and the regulation of differentiation and proliferation of several immune cells. Various inflammatory molecules regulated the secretion of IL-8 and IL-10, but the action of glutamate on the biosynthesis of cytokines is unknown. We have found that in peripheral blood lymphocytes glutamate at the concentrations within normal plasma levels (1 x 10(-5) M), as well as at lower concentration (0.3 x 10(-6) M) changes the secretion of immunosuppressive cytokine IL-10, whereas synthesis of proinflammatory chemokine, IL-8 did not changed significantly. Moreover, our results have shown that peripheral blood lymphocytes from patients with autoimmune thyroiditis release less IL-10 at both concentration of glutamate than peripheral blood lymphocytes from healthy persons. These data suggest that glutamate decrease the secretion of IL-10 by peripheral blood lymphocytes, especially in patients with autoimmune thyroiditis that may be responsible for prolongation of inflammation.

Growth hormone (GH) determinations by RIA and IFA during GH stimulation tests in children with short stature.

Growth hormone deficiency (GHD) as a syndrome comprises multiple pathogenetically distinct entities caused by disorders of secretion or peripherial action of growth hormone (GH). Confirmation of the diagnosis of GH deficiency in children is based on provocative testing for human growth hormone (hGH). Immunofunctional assay (IFA) allows quantitation of only biological active GH forms in circulation. The aim of this study was to compare the results of GH determinations by radioimmunoassay (RIA) and IFA, and to establish cut-off-levels for IFA in insulin tolerance test (ITT). We have investigated 32 children (30 males and 2 females) with short stature. All patients underwent insulin tolerance test (ITT), GH was measured in duplicate by radioimmunoassay (RIA, Seria) and IFA (DSL). Children with peak GH concentration below 10 ng/ml in ITT (by RIA) underwent arginin tolerance test (ATT, arginin 0,5 g/kg). The correlation between the results of growth hormone determination by radioimmunoassay and immunofunctional assay was excellent and cut-off-level in stimulatory test was established for the immunofunctional assay.