Keywords: Genetic study, Sleep disorders, Mutation HLA-DR2, HLA-DR14, HLADQB1
IntroductionFatal familial insomnia (FFI) is an extremely rare autosomal dominant inherited prion disease of the brain. It is almost always caused by a mutation to the protein PrPC, but can also develop spontaneously in patients with a non-inherited mutation variant called sporadic fatal insomnia (sFI). FFI has no known cure and involves progressively worsening insomnia, which leads to hallucinations, delirium, and confusional states like that of dementia [1]. The average survival span for patients diagnosed with FFI after the onset of symptoms is 18 months [2].
The mutated protein, called PrPSc, has been found in just 40 families worldwide, affecting about 100 people; if only one parent has the gene, the offspring have a 50% risk of inheriting it and developing the disease. With onset usually around middle age, it is essential that a potential patient be tested if they wish to avoid passing FFI on to their children. The first recorded case was an Italian man, who died in Venice in 1765 [3].
Materials and Methods
In this study, 140 patients with sleep disturbances and 160 healthy controls were studied. Peripheral blood samples from patients and parents with written permission control was prepared. After separation of serum, using Real Time-PCR technique of tRNA molecules were collected [4]. To isolate Neuroglial cells erythrocytes were precipitated from hydroxyethyl starch (HES) was used. At this stage, HES solution in ratio of 1 to 5 with the peripheral blood of patients and controls were mixed. After 60 minutes of incubation at room temperature, the supernatant was removed and centrifuged for 14 min at 400 Gera. The cells sediment with PBS (Phosphate Buffered Saline), pipetazh and slowly soluble carbohydrate ratio of 1 to 2 on ficole (Ficol) was pouredin the 480G was centrifuged for 34 minutes. Mono nuclear Neuroglial cells also are included, has a lower density than ficole and soon which they are based. The remaining erythrocytes has a molecular weight greater than ficole and deposited in test tubes [5].
The supernatant, which contained the mononuclear cells was removed, and the 400 Gera was centrifuged for 12 minutes. Finally, the sediment cell, the antibody and Neuroglial cells was added after 34 minutes incubation at 5 degree C, the cell mixture was passed from pillar LSMACS. Then the cells were washed with PBS and attached to the column LSMACSS pam Stem cell culture medium containing the transcription genes HLA-DQB1, HLA-DR2, HLA-DR14 and were kept [6].
To determine the purity of Neuroglial cells are extracted, flow cytometry was used.
For this purpose, approximately 4-5 * 103 Neuroglial cells were transfer red to 1.5 ml Eppendorf tube and then was centrifuged
at 2000 rpm for 7minutes a time. Remove the supernatant culture medium and there maining sediment, 100 μl of PBS buffer was added. After adding 5-10 μl CD4+ PE monoclonal anti body to the cell suspension for 60 min at 4 degree C, incubated and read immediately by flow cytometry. For example, rather than control anti body Neuroglial cells PE, IgG1 negative control solution was used [7].
Total RNA extraction procedure includes:
1. 1ml solution spilled Qiazolon cells, and slowly and carefully mixed and incubated at room temperature for 5 minutes. Then 200 μl chloroform solution to target mix, then transfer the micro tubes were added, and the shaker well was mixed for 15 seconds. The present mix for 4 minutes at room temperature and then incubated for 20 min at 4 degree C an was centrifuged at 13200 rpm era. Remove the upper phase product were transferred to a new micro tube and to the one times the volume of cold ethanol was added. The resulting mixture for 24 hours at -20 degree C were incubated [8].
2. Then for 45 min at 4 degree C an was centrifuged at 12000 rpm era. Remove the supernatant and the white precipitate, 1 ml of cold 75% ethanol was added to separate the sediment from micro tubes were vortex well. The resulting mixture for 20 min at 4 degree C an by the time we were centrifuged 12000 rpm. Ethanol and the sediment was removed and placed at room temperature until completely dry deposition. The precipitate was dissolved in 20 μl sterile water and at a later stage, the concentration of extracted RNA was determined [9].
To assessment the quality of mi-RNAs, the RT-PCR technique was used. The cDNA synthesis in reverse transcription reaction (RT) kit (Fermentas K1622) and 1μl oligoprimers18 (dT) was performed. Following the PCR reaction 2 μM dNTP, 1μg cDNA, Fermentas PCR buffer 1X, 0 / 75μM MgCl2, 1.25 U / μL Tag DNA at 95 degree C for 4min, 95 degree C for 30s, annealing temperature 58 degree C for 30s, and 72 degree C for 30 seconds, 35 cycles were performed. Then 1.5% agarose gel, the PCR product was dumped in wells after electrophores is with ethidium bromide staining and color were evaluated [10-12] (Figures 1 and 2).
Discussion and Conclusion
According to the results of sequencing the genome of patients with sleep disturbances, and the genetic mutations HLA-DR2, HLA-DR14, HLA-DQB1 genes found that about 100% of patients with sleep disturbances, they have this genetic mutation. Patients with sleep disturbances, unusual and frightening images in the process of sleep disturbances, experience. Lot epigenetic factors involved in sleep disturbances. But the most prominent factor to induce sleep disturbances, mutations is HLA-DR2, HLA-DR14, HLA-DQB1 genes. This genes can induce the birth and can also be induced in childhood and adulthood (Figure 3).