| Leber’s hereditary optic neuropathy (Leber’s Hereditary Optic Neuropathy, LHON) is a matrilineal genetic disease with mitochondrial dysfunctions, whose lesions are mainly about the optic disc macula bundle of nerve fibers. The main feature is acutely or sub-acutely loss of central vision with both eyes, and the onset of the crowd are mainly young or adult males. In1871, Theodor Leber, a German ophthalmologist, firstly reported and described the clinical features of LHON, after researching55patients of16families, while he recognized it as an independent genetic disease. This disease mostly performs as a optic neuritis in its early period, so it is also known as Leber optic neuritis; while after a long course of this disease, it will show as optic nerve atrophy in the later period, so it is also known as Leber optic nerve atrophy. The pathological mechanism of this disease has not been fully elucidated until now. Successively, some opinions are proposed such as X-linked recessive inheritance, the dominant inheritance of euchromosome, but neither can explain the characteristics and law of inheritance about LHON pedigrees. Later cytoplasmic genetics explains the clinical characteristics of LHON better. In1988, Wallace found the material basis of the cytoplasmic inheritance--mitochondrial DNA mutations, and firstly reported the first mitochondrial DNA mutations point11778which was related to LHON. After researching9pedigrees molecular biology, Wallace confirmed this point was in the ND4gene, and clearly declared that the mitochondrial DNA mutation was closely related with the disease, since then the research of LHON pathogenesis had been opened up a new direction. The complete human mitochondrial DNA (mtDNA) contains16,569pairs of circular DNA molecules, which can self-copy and be a independent genetic system of the nuclear chromosomes. It is different between the two basic groups which are composed of the mitochondrial DNA; one is a light chain, the other one is a heavy chain, and both of them have the function of genes encoding:encoding two kinds of rRNA,22kinds of tRNA and mRNA which contains13polypeptide chains(cytochrome-B, cytochrome-C, subunits of oxidase Ⅰ, Ⅱ, Ⅲ, ATPase subunit-6,-8and7subunits of the NADH dehydrogenase:ND1, ND2, ND3, ND4, ND5, ND6, ND4L). Thus, the mitochondrial DNA coding gene mutation will inevitably bring some barriers to the process of cell’s oxidation and phosphorylation, and lead to less of energy for cells. On the basis of Wallace’s studies, in1991, Huoponen K, etc. studied3Finnish families and found the mutation point3460in the ND1gene; Johns DR, etc. studied14families and found found the mutation point14484in the ND6gene [2-3]. G11778A, G3460A, T14484C are familiar to a number of scholars as the primary mutation points of LHON. Complex I gene sequences in the encoded respiratory chain are closely related to95%of the clinical LHON cases, and in China, LHON patients mostly suffer the mutation point11778(90.9%), in contrast, the3460and14484mutations take low rate. The reason that these three mutations is not a crowd polymorphism but the primary point mutations (primary mutation), is that, their separate existences can result in patients with LHON clinical phenotype, the basic group of mutation points take less and conservative part in the evolution of coded respiratory chain of amino acids and they do not exist in normal populations. It has been confirmed, the single base mutations of a variety of mitochondrial DNA are related with LHON pathogenesis, and the risk factors causing blindness include three kinds of mitochondrial DNA of primary mutations (3460,11778,14484), but there are many questions about the gender bias of LHON, the incomplete penetrance and its pathological mechanisms. For example, Zhang Qingjiong etc. has studied and found that not all affected pedigree members of Chinese people carry the11778points, and52.3%male and13.8%female have LHON clinical features. Further studies showed that less than30LHON-related mitochondrial secondary mutation points, the Chinese people include3394,9438,4216. But the theory of secondary mutation points, the nuclear genetic background, haplotype differences and so on, can not fully explain the etiology of LHON, clinical manifestations, and family members of clinical characteristics.Because the etiology and exact pathogenesis is not fully elucidated, there is no effective control measures until now. Plenty of patients with severe loss of visual function need urgent medical treatment. And more people with high-risk genetics problems are looking forward to effective preventive measures. From studies on LHON, there is incomplete penetrance and penetrance differences among a considerable number of families, and this suggest that the incidence of LHON disease process, only the existence of mitochondrial DNA mutation is not sufficient to lead to the mutation point and basic group carriers of LHON family onset, there may be a "second hit" procedure. If the procedure happened after the birth of the pedigree members, we can find measures to prevent the onset; if the procedure happened before the birth, it is also possible to find an effective means to treatment the disease. According to the researches, the apoptosis process of retinal ganglion cell of LHON patients may be related to the signal transduction pathways of Fas/FasL mediated apoptosis. In the signal transduction pathways of Fas/FasL mediated apoptosis, Caspase-8is the initiated factor of apoptosis initiation, which also participates in the process of Fas mediated or non-Fas mediated apoptosis. Therefore, we speculated that the aforementioned aspects of the "second hit" may be related to the Caspase-8gene expression, while in the process of transcription, translation and expression, the methylation status of Caspase-8gene plays a very important role in this process.Study Purpose:1After doing ophthalmic examination and molecular genetics testing for a big LHON pedigree from Jiangxi Province, China, and understanding the form of the family disease patients with mitochondrial DNA mutation point and their genetic characteristics, it is confirmed that the existence of "second hit theory" in the "first hit" is true. It will be analyzed the3primary mutation points, in order to explore the pedigrees whether carry any currently known mitochondrial DNA primary mutation point or not.2Detect LHON carriers, patients with LHON, family members without LHON and healthy people (not belong to this family), and research their Caspase-8gene methylation or demethylation state, Caspase-8protein expression in the pedigrees, in order to discuss that if there are differences of methylation status among these group of people or not, and whether it is a factor of the pathogenesis of LHON.3According to previous research results, the experiment objects are divided into four groups:family members with carrying LHON mutation points but not incidence, LHON patients, family members without carrying the mutation points and the normal compared group (not have LHON and not belong to LHON family). The reactivity of sFasL apoptosis for the peripheral blood lymphocytes will be explored and the function will be discussed if z-VAD-fmk can suppress the apoptosis rate of peripheral blood mononuclear cell.Research Methods:1Ophthalmic examination:Firstly the clinical diagnosis standard will be established, then proceed some contrasted researches on visual functions such as vision, intraocular pressure, slit-lamp biomicroscopy, improvement of quantitative Amlser box, FM100-Hue chromometry, Humphrey750perimetry and so on. After pupil dilation, retinal mirror, multifunctional fundus image processing system and OCT3will be used to detect the changes of organizational structures in choroid and retina. It is aimed to investigate that whether the pathogenesis is a process from quantitative change to qualitative change, or full/none phenomenon, in order to support the "second hit" theory.2Follow the principle of informed consent, extract peripheral venous blood from LHON family members and normal family members, isolated lymphocytes, isolate lymph cells and peripheral blood mononuclear cells (PBMC, including monocytes and lymphocytes), prepare DNA, amplify PCR, test the sequence of product purification, identify the mitochondrial DNA mutation points, G3460A, G11778A, etc..3According the experimental results during the early period, the research objects on next step are divided into:LHON family member patients, LHON family member carriers, normal family members, other family members without LHON mutation point genes. According to the selection criteria for the above four groups, perform on peripheral blood collection once again, isolate the peripheral blood mononuclear cells (PBMC), extract the total cellular protein, prepare cDNA, use the method of western-blot to detect the expression amount of Caspase-8protein, and compare with four group of persons in order to check whether differences exist among Caspase-8mRNA or not.4Following steps of the former experiment, extract the peripheral blood mononuclear cell from four groups of people, extract the genomic DNA, the genomic DNA with bisulfite modification, the PCR testing with methylation of the Caspase-8gene, and analyze the results about the differences on Caspase-8gene methylation degree among four groups.5Grouping by the researching results in the early period:paitents of Leber Hereditary Optic Neuropathy, carriers of LHON mutation points, family members without LHON genes and healthy people not in this family. Respectively test the apoptotic rate of peripheral blood mononuclear cells for the four groups after the sFasL induction apoptosis treatment, and finally compare their differences.6Use Z-VAD-fmk pre-handle the peripheral blood mononuclear cells of leber hereditary optic neuropathy patients, detect and analyze the apoptotic rate of cells after sFasL induction between the pretreatment group and the untreated group, and finally compare their differences.Research results:1. In the family there are24mutation point carriers, including11patients. After the onset the ranges of their visions start from light perception to0.1, and10patients of them suffer visual impairment very severely. The10LHON patients’mean age of onset is24.36years old, and the largest age is50years, while the minimum age is8years old. While people carrying the mutation points but have not come on yet whose average age is40.38, of which the maximum age is72years old, and the minimum age is5years. There are statistics differences (t=2.102, P=0.049) between the ages of LHON patients and mutation point carriers. Family patients has the common characteristic that is with severe visual impairment, but other eye diseases and deafness, ataxia and other nervous system complications are not found. Only in one case the fallen of binocular vision is separated by one week, while the remaining two patients suffer vision loss of both eyes simultaneously with eye pain and other discomfort. But the visions of all the patients in this present study pedigree are lower than0.01, and so far no visual function recovery has been found. Binocular fundus fluorescein angiography examination shows that:the capillary on binocular optic disks has never been found with fluorescence filling, and only on the edge of disc there is some fluorescent staining during the later period, and no fluorescence shielding or retinal vascular leakage. Some phenomena, such as typical optic nerve papilla shallow telangiectasia during early period or retinal vascular tortuosity and dilatation have not shown, due to optic nerve atrophy in such a long disease course.2Among72maternal family members, there are24persons who carry the G11778A and T14502C mutation points. There are10males and1female in11LHON patients with the ratio male/female=10:1, and the other13carriers has not come on LHON. The other family members and20research objects in normal contrast groups do not carry the mutation points. All the members were not found having G3460A and T14484C, two mutation points.3It has been found through the Western-blot method of Caspase-8protein detecting the peripheral blood mononuclear cells (PBMC) of four groups:LHON family member patients, LHON family member carriers, normal family members, other family members without LHON mutation point genes, that the Caspase-8protein expression level of LHON patients raises up obviously than the other three groups, and these differences have statistical significance (F=16.124,P=0.000)4Doing researches of Caspase-8gene methylation degree on the four groups, the results display:the positive rate of Caspase-8gene unmethylation of LHON pedigree members is significantly higher than that mutation genes carrier, family members without LHON mutation genes, and healthy people who not belong to this pedigree (X2=20.466, P=0.000)5The research about sFasL induced functions for the apoptosis of mononuclear cells:Using Annexin V-FITC and PI double staining flow cytometry to test the level of cell apoptosis after sFasL induction. The results show that after using sFasL induced, the rates of apoptosis in mononuclear cells of each group have increased compared with the negative contrasted samples(t=18.877,P=0.000). Compared to the onset members in the family, the family members who not carry the mutation point and normal people, the mononuclear cell apoptosis rate of the Leber’s hereditary optic neuropathy sick members of this pedigrees significantly increase (F=3.024, P=0.036). But except the group of the sick members, the other three groups of objects, have no significant statistic differences in apoptosis rate (P>0.05)6Use z-VAD-fmk for pre-handling and then use sFasL for apoptosis induction to all the peripheral blood mononuclear cells of leber hereditary optic neuropathy patients. It has been found that:after the induction of sFasL, the apoptosis rate of patients has reached0.139±0.040, and compared with it, after the the intervention of the appropriate dose of z-VAD-fmk, the apoptosis rate of the same sample cells significantly gets lower. In the intervention group and non-intervention group, the apoptosis rate differences are statistically significant (t=3.904, P=0.003)Conclusion:1The family patients mostly suffer severe visual impairment; the family members carry the mutation points11778and14502; all patients had no visual recovery until now, all those situations are identical with the reports about carrying 11778mutation point abroad:the most serious visual impairment of patients, rare recovery or improvement, poor prognosis. But the domestic researchers reported that in Chinese11778mutation patients52.1%of their vision is between0.01and0.1,30.3%is higher than0.1, eventually recovering rate can reach up to6.8%. Compared with the researches above, the clinical presentation and prognosis of this present study pedigree are worse than that of only carrying11778pedigrees. Therefore,11778mutation point may work together and have some cooperation with14502.2The research results show that the average onset age of the present pedigree is24.36years, except two cases that older than35years, the onset ages of remaining patients are identical with report abroad,15-35years old. The patients of this pedigree get a sex ratio Male:Female=10:1, which exists severe gender bias. One of the important disease characteristic of LHON is incomplete penetrance, this family is45.8%. A lot of mutation points carriers have not come on LHON even their ages are significantly older than the average age of onset. This illustrates that the existence of mitochondrial DNA primary mutation is not enough, as the necessary condition "first hit" is not adequate and it may need "second hit". At the same time" second hit" and "first hit" should have sufficient strong synergistic effect.3As to Caspase-8which participates in mediated apoptosis pathway of Fas and not Fas, the expression of protein in peripheral blood mononuclear cells of the family patients is significantly higher than that of carriers and the normal population. It has been researched that, compared with normal cells, the original LHON pathological mutant cells are more sensitive to Fas medium mediated apoptosis. It is proved that, in this pedigree, the abnormal elevation of the protein expression of Caspase-8of LHON patients may have some relationships with the easier happenness of cell apoptosis when these mitochondrial cells carrying DNA mutation points are in this pathological environment.4The positive rate of Caspase-8gene methylation in family LHON members is significantly higher than that of mutation gene carriers but not incidence, family members without mutation gene and normal persions of other families. It illustrates that the methylation status of Caspase-8gene influences gene transcription efficiency, thus this may be an important reason why family members carrying LHON mutation point will suffer it.5The sensitivity of the peripheral blood mononuclear cells of LHON sick family members for the sFasL apoptosis induction is significantly higher than the mutant gene carrying members but not the incidence, family members without carry ing mutation genes and healthy people outside the family. Combined with the "Conclusion3", it can be found that on the foundation that the same family members carry the same mitochondrial DNA mutation points, compared with carriers, not only the unmethylation positive rate of Caspase-8genes of LHON patients’mononuclear cells significantly increases, but also in the same apoptosis-inducing treatment condition, the apoptosis rate of patient group was higher, and therefore it can be inferred that the Caspase-8gene methylation status may make mutation points carriers onset through the promoted apoptosis sensitivity.6As to z-VAD-fmk, there are some protected functions to the apoptosis procedure of the peripheral blood mononuclear cells inducted by sFasL induction of LHON patients. This effect is outstanding, and it can significantly reduce the rate of cell apoptosis in the face of apoptosis induced treatment. |
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