Studying On Silkworm Lemon (Lem) Mutant As The Model Of Human Sepiapterin Reductase Deficiency

Animal models of human diseases refer to the animals with simulated manifestations of human diseases established in various medical scientific researches,which are the link between basic and clinical researches.By constructing animal models of various diseases,it is possible to investigate the functions of some diseases-related genes,and reveal the mechanism of disease occurrence and development,which is an advantage for drug screening,drug efficacy evaluation and the development of disease diagnosis technology and treatment.Therefore,animal models of human diseases play an important role in the field of medical research.Human sepiapterin reductase deficiency is an autosomal recessive disorder caused by the mutation of SPR gene.The disease always misses the best treatment time because it is difficult to be detected in neonatal genetic screening and is misdiagnosed easily as other diseases.In addition,the therapeutic method of sepiapterin reductase deficiency is restricted.Therefore,it is necessary to conduct in-depth researches on sepiapterin reductase deficiency in order to establish more methods for accurate diagnosis and develop drugs that are much more effective.Silkworm?Bombyx mori?,an important economic insect,is a classical genetic model organism.Silkworm has the advantages of clear genetic background,moderate size,easy feeding,short life cycle and high reproductive rate.Except that,it has a 58%homology rate with human genes and many homologous genes related to diseases.Therefore,silkworm has potentials to act as an animal model of human diseases.In this study,we explored whether lem mutant could be as the animal model of sepiapterin reductase deficiency.The results obtained are as follows:1.Phylogenetic analysis of the pathogenic gene SPR of sepiapterin reductase deficiencyTo understand the conservation of the SPR gene between human and common model animals,we performed a phylogenetic analysis of the SPR gene.We downloaded the protein sequences of SPR gene in Homo sapiens,Macaca mulatta,Mus musculus,Rattus norvegicus,Xenopus tropicalis,Danio rerio,Drosophila melanogaster,Aedes aegypti and Bombyx mori from NCBI and constructed the phylogenetic tree with MEGA 5.0software.It found that the SPR gene is relatively conservative in different evolutionary classes,and the SPR gene of Bombyx mori and Operophtera brumata are the most conservative.2.Cloning and expression analysis of BmSPR gene and related genes in lem mutantHuman sepiapterin reductase deficiency is caused by the mutation of SPR gene.Through molecular cloning experiments,it found that the BmSPR gene of lem mutant has a point mutation,which lead to the premature translation termination and thereby the deletion of last five amino acids at the carbon terminus,which is similar to the pathogenesis of sepiapterin reductase deficiency.To understand the expression characteristics of BmSPR gene,we investigated the expression profile of the BmSPR gene in lem mutant.The results showed that the BmSPR gene expresses in each period(from 1^st to 5^th instar larvae).Among them,the expression level of BmSPR gene in lem mutant is higher than that in other periods during the day 1of 2^nd instar larvae,which indicates that the BmSPR gene is more important in the early stage of silkworm larvae,and it is consistent with the clinical features of sepiapterin reductase deficiency in infancy.We also investigated the expression of some key genes of the BH4 pathway including BmGTPCH I,BmPTPS,BmDHFR,BmPAH,and BmTH in lem mutant during early stages.The results showed that the expression changes of these five genes is reasonable when the BmSPR gene mutates,which also confirmed the conservation of BH4 pathway in silkworm and human.3.Phenotypic investigation of the lem mutant as the animal model of sepiapterin reductase deficiencyWhen constructing animal models of human diseases,it is necessary to consider whether the selected animal model has a phenotype to observe easily.We observed that the lem mutant is yellow-colored and has no marking.Therefore,we investigated the relationship between this phenotype and BmSPR gene through the multi-strains validation experiment.We found that the BmSPR gene of the nine yellow-body color strains has the same point mutation,indicating that the yellow body color is related to BmSPR gene,and this phenotype might favor lem mutant as the model of sepiapterin reductase deficiency.The clinical manifestations of sepiapterin reductase deficiency include stunting,dyskinesia,dystonia and so on.Therefore,we investigated the body weight and body length of lem mutant and wildtype strain Dazao.The results showed that the lem mutants are larger and heavier than the wildtype,which means BmSPR is not negative for the body weight and body length of lem mutant.We also detected the content of blood glucose,however,no significant difference was found between lem mutant and Dazao,which means that the BmSPR gene has less effect on the development of silkworm.We explored the mobility of lem mutant through the mulberry leaf-luring test and the turnover test.It found that lem mutant group spent less time to arrive the mulberry leaves than the control group,and the time for the turnover test between lem mutant and Dazao had no significant difference,which means that the mutation of BmSPR gene had no negative effect on body development and behavioral abilities of lem mutant.4.Biochemical feature evaluation of the lem mutant as the animal model of sepiapterin reductase deficiencySepiapterin reductase deficiency does not show hyperphenylalaninemia,which is one of the diagnostic features.Therefore,we compared the phenylalanine content in the blood of lem mutant and Dazao,and found that the content of phenylalanine had no significant difference between lem mutant and Dazao,which was consistent with sepiapterin reductase deficiency.The content of dopamine and serotonin in cerebrospinal fluid of the patient with sepiapterin reductase deficiency is significantly low,which is one of the important indicators for clinical diagnosis.We used UPLC technology to detect the content of these two neurotransmitters in the brains of lem mutant and Dazao,and found that the content of dopamine and serotonin in lem mutant was significantly lower than that in Dazao,which accords with sepiapterin reductase deficiency.Sepiapterin reductase deficiency shows increased level of neopterin.We detected the content of neopterin in the brains of lem mutant and Dazao by UPLC.It found that the content of neopterin in the lem mutant was significantly higher than that in Dazao,which is consistent with the performance of sepiapterin reductase deficiency.5.The biochemical change after feeding with L-dopa in lem mutant was consistent with the therapeutic effect of sepiapterin reductase deficiencyL-dopa in combination with carbidopa is the main therapy method to correct CNS dopamine deficiency.Therefore,we fed lem mutant with three concentrations of these two compounds.The results showed that the dopamine content in the brains of three experimental group was significantly higher than that in the control group,which is consistent with the therapeutic effect of sepiapterin reductase deficiency at biochemical level.However,there was no significant change in phenotype,which might be because of that the concentration of L-dopa and carbidopa was too low to form enough dopamine for melanin synthesis or the content of synthesized melanin is too low to show significant body color changes.In summary,based on the current experimental results,we initially believe that the lem mutant meets the requirements as the animal model of human sepiapterin reductase deficiency at the genetic and biochemical levels,which provides potential help in the in-depth study of the therapeutic method of this disease and the development of new drugs.