Identification Of Pathogenic Mutations In Two Rare Diseases And Investigation Into The Pathogenesis Of TSC

Rare diseases are kinds of diseases with low incidence,which are chronic and serious and often endanger life.According to the definition of The World Health Organization(WHO),rare diseases are that the number of patients with diseases accounts for 0.65‰ ～ 1‰ of the total population.Wolcott-Rallison syndrome(WRS,OMIM: 226980)is a rare autosomal recessive inheritance disorder caused by mutations in the eukaryotic translation initiation factor 2 alpha kinase 3(EIF2AK3)which encodes the PKR-like endoplasmic reticulum kinase(PERK).Defect in EIF2AK3 results in a permanent diabetes in the early infancy or newborn period,a tendency to develop skeletal fractures and other associated disorders such as severe liver and renal dysfunction.Tuberous sclerosis(TSC)is an autosomal dominant genetic disease that involves multiple tissues and organs,such as kidney,heart,lung,skin,brain and other organs,caused by the defect or loss of functions of TSC1 and TSC2 encoded by mutated TSC1 or TSC2 respectively.The incidence of TSC is independent of country,ethnicity,and gender.Epidemiological studies have shown that one in every 6,000 to 10,000 people worldwide suffers with TSC,and there are about 2 million TSC patients worldwide,of which,more than 80%of the patients will suffer from bilateral multiple angiomatosis,which is also the main cause of death in TSC patients.So far,there is no effective targeted drug available for TSC in the world.The fine clarification and deep understanding of TSC pathogenesis is the most crucial for the development of effective and precise interventions for TSC.In this study,families with these two rare diseases were recruited individually.The main objectives are to identify causative mutations of the patients from one family with WRS using whole exome sequencing(WES)and Sanger sequencing,analyses the all reported mutations to illustrated the relationship between the mutations and the phenotypes of WRS patients.This thesis mainly focuses on the studies of two mutations related to two families with TSC.Identification of the mutations in either TSC1 or TSC2 was carried out using whole exome sequencing and Sanger sequencing,then phenotypic analyses of the mutated genes and investigation into mechanisms underlying TSC caused by either TSC1 or TSC2 mutations in two families were conducted using cell lines expressing mutated TSC1 or TSC2 and human skin fibroblasts isolated from the patient.The results and findings provide a new insight of some novel biological functions of TSC1 and TSC2,and reveal novel mechanisms underlying TSC caused by either TSC1 or TSC2 mutation which yielding new information with scientific significance contributing to the precision medicine for TSC therapy.The Ph D thesis is mainly divided into five parts as the followings:Part I: Mutation identification of WRS patients and bioinformatic analyses of WRS.Whole exome sequencing was performed using samples of the whole peripheral blood from the patients in the family with WRS.Bioinformatic analysis of the two sets of data from this family,two novel compound heterozygous EIF2AK3 mutations were identified.Both mutation c.2818C>T and c.2980G>C are missense mutations.The Sanger sequencing showed that the mutation c.2818C>T was from their father,and the mutation c.2980G>C was from their mother.Both mutations were located at the kinase domain of PERK,resided at highly conservative region of the protein in particular,suggesting both mutations are pathogenic.Based on the Human Gene Mutation Database(HGMD),this study systematically analyzed the reported mutations in EIF2AK3 to illustrate the relationship between the mutations and the phenotypes of WRS patients,which should be useful to precision medicine.Part II: Bioinformatic analyses and mutation identification of TSC patients.The samples of peripheral blood of two patients and members from two independent families with TSC were collected after clinicians had diagnosed them as TSC through a complete set of physiological and biochemical examinations and analyses.WES was performed using the whole peripheral blood of the probands from those two families.Bioinformatic analyses of the two sets of data from two families,and two mutations were identified,one was a deletion mutation c.2509＿2512del(p.Asn837Valfs*11)located in the TSC1,which has been reported,but the pathogenicity and the mechanism underlying TSC caused by the mutation have not been investigated yet,and the other one was a deletion mutation c.1113 del G(p.Gln371Hisfs*18)located in the TSC2.The mutation c.1113 del G is a novel mutation which has not been reported before.The Sanger sequencing showed that the mother and the son of the proband with the mutation c.2509＿2512del(p.Asn837Valfs*11)were carriers of the mutation,but the proband’s wife had no mutation in either TSC1 or TSC2.The daughter of the proband with mutation c.1113 del G(p.Gln371Hisfs*18)was the mutation carrier.Part III: Effect of the mutations on cell biological phenotypes and related signal pathways.We isolated and generated human skin fibroblasts(HSFs)from a patient carrying the deletion mutation c.1113 del G(p.Gln371Hisfs*18)and two healthy volunteers respectively.The results of cell proliferation assays showed that the proliferation rate of mutant HSFs was significantly higher than that of HSFs isolated from healthy volunteers.Results of cell cycle assays indicated that the number of cells in the M phase in mutant HSFs was significantly greater than that of HSFs isolated from healthy volunteers.Results of apoptosis assays demonstrated that the apoptosis rate of mutant HSFs was lower than that of HSFs isolated from healthy volunteers.These phenotypic analyses suggested that the deletion mutation c.1113 del G(p.Gln371Hisfs*18)in TSC2 affects cell proliferation.Further,we quantified TSC2 expression at both m RNA and protein levels.RT-q PCR results showed that the m RNA expression level of TSC2 in mutant HSFs was reduced by about half compared with that in HSFs from healthy volunteers.Western blot,immunofluorescence and flow cytometry assays also indicated that the mutation c.1113 del G(p.Gln371Hisfs*18)reduced the expression of TSC2 in HSFs.The m RNA quantification using RT-q PCR demonstrated the deletion in TSC2 resulted in increased expression of VEGF,m TOR and HIF-1α downstream of TSC2.The m TORC1 pathway was excessively activated in HSFs with the mutation c.1113 del G(p.Gln371Hisfs*18)due to the decrease of TSC2 expression.When the wild-type TSC2 was transformed in the HSFs isolated from the patient,the activity of m TORC1 pathway was rescued.Co-IP and non-denaturing gel experiments showed that both the mutations c.2509＿2512del and c.1113 del G were unable to form a functional complex,leading its inability to regulate the activity of m TORC1 pathway.Part IV: The regulation of the expression of m TOR by TSC complex through mi R-199b-3p.To explore the molecular mechanism underlying the relationship between TSC2 and m TOR in term of their expression in HSFs.In mi RBase database,we found that mi R-199b-3p may regulate the expression of m TOR.Analysis of the m RNA in HSFs using RT-q PCR showed that the expression of mi R-199b-3p in mutant HSFs was significantly lower than that in HSFs from healthy volunteers.The results of western blot demonstrated that the expression of m TOR in mutant HSFs increased significantly with comparison of that in HSFs from healthy volunteers.The dual-luciferase reporter assays suggested that mi R-199b-3p can directly bind to the 3’UTR region of m RNA of m TOR so that regulates the expression of m TOR at the post-transcriptional level.In HEK293 T cells,both RT-q PCR and western blot analyses demonstrated that mi R-199b-3p was negatively correlated with m TOR protein expression.In order to further explore the relationship between TSC1/2 and mi R-199b-3p,the knockdown and overexpression of TSC1 or TSC2 was performed,the quantification assays showed the expression of mi R-199b-3p in cells was significantly decreased,and the expression of m TOR in cells was significantly increased in the cells knocked down either TSC1 or TSC2,the expression of mi R-199b-3p in cells was significantly increased,and the expression of m TOR in cells was significantly decreased in the cells overexpression either TSC1 or TSC2.These results suggested that the TSC complex is involved in the negative regulation of the expression of intracellular mi R-199b-3p,and then causes the change of m TOR protein expression,it is revealed for the first time that TSC1/TSC2\mi R-199b-3p\m TOR in the form of an axis participates in the pathophysiological process of Tuberous Sclerosis,this is a novel mechanism of Tuberous Sclerosis causing by TSC1 or TSC2 mutations.Part V : Molecular mechanism underlying TSC1 and TSC2 regulating glycolysis.The results of the glycolysis capacity assays showed the glycolysis capacity of HSFs from patients was significantly reduced compared with that of HSFs from healthy volunteers.The glycolysis of cells with TSC1 knockdown was increased,but the glycolysis of cells with TSC2 knockdown was decreased.Western blot analysis showed that the expression of HIF-1α,HK2 and PKM2,the key proteins which regulate glycolysis,increased after TSC1 knockdown.The expression levels of HIF-1α and HK2 also increased after TSC2 knockdown,while the expression levels of PKM2 were decreased.When overexpressed TSC1,the m RNA levels of HIF-1α,HK2 and PKM2 were significantly increased,and the western blot demonstrated the protein level of PKM2 was also significantly increased,the expression level of HIF-1α was significantly decreased.However,the m RNA levels of HIF-1α,HK2 and PKM2 had no significant change in the cells with the overexpression of TSC2.The expression levels of HK2 and PKM2 had no significant change,corresponding to their m RNA expression,but the expression level of HIF-1α decreased significantly in the cells with the overexpression of TSC2.The enzyme activity assays indicated the overexpression of TSC1 and TSC2 had no significant effect on the activity of HK,while the activity of PK was significantly increased.The lactate dehydrogenase(LDH)activity was decreased in cells with the overexpression of TSC1.The LDH activity increased in cells with the overexpression TSC2.This was consistent with previous experiments on glycolysis activities.All results above suggested that the TSC1 and TSC2 might regulate glycolysis by regulating the activity and the expression of key enzymes in glycolysis,all results above revealed a novel function of TSC1 and TSC2,which involves glycolysis,and defected functions of TSC1 or TSC2 mutants caused by TSC1 or TSC2 mutation will introduce abnormal glycolysis,leading to the pathophysiological process of TSC.In summary,the present study identified two novel mutations in two patients from a WRS family,which provides useful information guiding precise WRS diagnosis and getting novel and effective interventions.At the same time,we identified two mutations in two patients with TSC from two independent families and found a novel mutation,enriching the TSC mutation database.The pathogenesis of the mutations was experimentally verified,which provides useful information guiding the precise TSC diagnosis at molecular level and generation of novel and effective interventions.We first revealed that the TSC complex which formed by TSC1 and TSC2 not only regulates the phosphorylation of m TOR,but also regulates the expression of m TOR through mi R-199b-3p,indicating for the first time that TSC1/TSC2\mi R-199b-3p\m TOR in the form of an axis participates in the cell physiological processes downstream of TSC1/TSC2 complex.Furthermore,the results given by serials of experiments revealed another novel molecular cell mechanism underlying TSC.The regulation paradigms of glycolysis by TSC1 and TSC2 are different,which may explain the difference in the symptoms or clinical presentations and the severity of TSC caused by TSC1 mutation and TSC2 mutation respectively.All information yielded from this project reveals novel mechanisms underlying TSC caused by either TSC1 mutation or TSC2 mutation and provides some useful novel information for precise medicine for TSC.Results and findings of the project also illustrate novel physiological functions of TSC1 and TSC2,extending our understanding of TSC1 and TSC2 biological functions.