A Zebrafish Model For Human Congenital Erythrocyte Porphyria And Its Phenotypic Heterogeneity

Porphyria is a type of disease caused by defects in the heme biosynthesis.Different enzyme defects in the heme biosynthesis lead to different porphyria.These porphyria are divided into livers porphyria and erythrocyte porphyria according to the organ accumulated by porphyrin.Congenital erythrocyte porphyria(CEP)caused by defects in Uroporphyrinogen ? synthase(UROS)belongs to erythrocyte porphyria.Human congenital erythrocyte porphyria patients have phenotypes such as skin light sensitivity,hemolytic anemia,hepatosplenomegaly and porphyrin urine,and these phenotypes are highly variable.Even the UROS with the same mutation,the clinical phenotype is not fixed,this phenomenon is called phenotypic heterogeneity.At present,there is no clear understanding of the specific mechanism of phenotypic heterogeneity,and there is no report on the zebrafish disease model for CEP.Here,we knock-out the uros-/-in zebrafish by CRISPR-Cas9,and found that the tiros-/-mutant showed red fluorescence under ultraviolet light and was consistent with the uros-/-expression,indicating that red fluorescence was due to the accumulation of coproporphyrin ? and uroporphyrin ? is caused.The uros-/-mutant is lethal,and it is more likely to die under normal light conditions than under dark conditions.The uros-/-mutant also exhibits a reduced phenotype of heme and hemoglobin.Through sequence alignment analysis,we found that uros-/-is highly conserved.Zebrafish uros mRNA and human UROS mRNA can rescue the reduced phenotype of uros-/-mutant hemoglobin,but it can not reduce the accumulation of c coproporphyrin ? and uroporphyrin ?.Exogenous hemin does not affect the phenotype of the uros-/-mutant.The transcriptome sequencing results and the real-time PCR showed that tspo(translocator protein)was up-regulated by porphyrin activation,while TSPO is a very conserved,which itself is able to bind porphyrin and even degrade protoporphyrin ? in bacteria[1].Therefore,we treated the WT and uros-/-mutant zebrafish with the TSPO inhibitor PK 11195 and found that the heme synthesis rate-limiting enzyme alasl was up-regulated,and the hemoglobin expression was improved in the uros-/-mutant.Next,we constructed the tspo mutant by CRISPR-Cas9 and found that the expression of the alasl,heme synthesis rate-limiting enzyme,is also up-regulated in the tspo mutant,but the tspo mutant phenotype is consistent with WT and can grow normally.We constructed a transgenic zebrafish Tg(hsp70l:tspo;CG)that can overexpressed tspo.Overexpression of tspo in WT or uros-/-mutants did not alter hemoglobin expression,but leads to up-regulation of the heme synthesis rate-limiting enzyme alasl.Moreover,continued overexpression of tspo in the uros-/-mutant prolongs the uros-/-mutant death time.In summary,we believe that the uros-/-mutant can be used as a model for human congenital erythrocyte porphyria,which lays a foundation for subsequent pathological mechanism research and drug screening.And we found that tspo can affect heme synthesis and slow down the toxic effects of porphyrins,determine the direction for subsequent phenotypic heterogeneity studies.