The Molecular Mechanism Of MYH13 Gene Mutation With Aniridia And Study On CMY2Q Mouse Model

PART 1 The molecular mechanism of MYH13 gene mutation with aniridiaAniridia is a kind of eye disease with a bilateral congenital absence or malformation of the irides,and patients tend to visual acuity decreased and nystagmus.Disease usually occurred in childhood and can be inherited in an autosomal dominant（AD）.Our research group collected a large pedigree with aniridia,9 of 24 were patients,who presented with complete absence of iris,accompanied by ptosis and cataract.We found a missense mutation in mysion heavy chain13(MYH13,c.542C>T（p.Ser180Tyr）using whole-exon sequencing technology.We constructed mutant plasmid and transfected it into HEK293 cells,the results showed the RNA expression of MYH13 and PAX6 were higher than control group,but COXIV expression was declined which meant mitochondrial was damaged in mice.Using transmission electron microscope,we observed the severe vacuolization of mitochondria in cells which were transfested with mutant plasmid.ATP assay result showed that the level of ATP in the mutant group was lower than that in the normal group;molecular model MYH13 showed that the wild type was-181.80kcal/mol and the Ser180 Tyr mutant was-161.76kcal/mol,which indicated that the binding ability of ATP was reduced after the Ser180 Tyr mutation;All of these results suggesting that missense mutation in MYH13 can affect the morphology and the function of mitochondrial.The expression of MYH13 in mice were expressed in the extraocular muscles,spleen and testis;Immunohistochemical staining of mouse eyes also showed that MYH13 protein was mainly expressed in extraocular muscles.At the same time,we constructed MYH13 knock-in and knock-out mice model by genetic engineeringmethod.Analysis of phenotype of knock-in mice showed that wild-type and heterozygous mice were not significantly different in size,weight,exercise capacity and eyes;but the the ratio of three genotypes about born mice was 3:5:1,which against the genetic law of Mendel.We need further observation and analysis for this phenomenon.The results of blood biochemical indexes showed that there was no significant difference in blood glucose,blood lipid and renal function in the three genotypes of mice.We hardly found difference about HE staining of three genotypes of mice,which need to be futher observed.PART 2 Study on Dhtkd1^Tyr486*knock-in mouse model with PTC124Charcot-Marie-Tooth disease called as hereditary sensory motor neuropathy,peripheral neuropathy is a genetic disease of a single gene with obvious clinical and genetic heterogeneity,which the incidence rate is about 1/2500.The genetic mode of the disease includes autosomal dominant inheritance,autosomal recessive inheritance and X linkage dominant or recessive inheritance.Our research group collected a CMT pedigree in Gaomi,Shandong,found a nonsense mutation of dehydrogenase E1 and transketolase domain containing 1（DHTKD1）by whole genome scan,linkage analysis and candidate genes analysis.The CMT caused by this mutation was named CMT2 Q.Using the method of site directed mutagenesis of Dhtkd1 gene mutation in mice in the same position,we successed to obtain a knock in mouse model with Dhtkd1 mutation of Tyr486*.Previous results of the phenotypic analysis showed that the features of mouse model was similar to the CMT2 Q type,just appeared in the micro structure and expression level.So,we found the expression of Dgkg gene was up-regulated,while Lpin2 gene was down-regulated in homo mice using RNA chip technology.At the same time,blood biochemical indicators showed decline in TG and TCHO of homozygote（P <0.05）may improve lipid metabolism to repair energy shortage which caused by low level of Dhtkd1.So we treated knock-in mice with PTC124,found the RNA level of Dhtkd1 and Dgkg were up-regulated,while the expression of Lpin2 was down-regulated（p<0.05）after two week;Protein levels suggested that the Dhtkd1 protein could be translated using 30ug/g/d,in vitro test results showed that the increase of diacylglycerol and decrease of phosphatidic（P<0.05）with 30ug/g/d concentration in mice serum,which suggested that it was effective to treat Dhtkd1^Tyr486*knock mice model with PTC124.