Clinical Characteristics,mutation Analysis And The Establishment Of IPS Cells Line With Liver Organoid Derived From Peripheral Blood Mononuclear Cells Of Urea Cycle Disorder Patients

BackgroundUrea Cycle Disorder(UCD)is an inborn error disease which caused by mutations in any one of the six enzymes involved in the Urea Cycle.The incidence of UCD varies from 1:8,000 to 1:45,000,as reported in the literature.Except for the X-linked inheritance of Ornithine Transcarbamylase Deficiency(OTCD),the other five are all autosomal recessive inheritance.According to the onset time,UCD can be divided into early onset and late onset,which is clinically manifested as the disease process of hyperaminemia.The clinical symptoms of early-onset UCD are extremely serious due to severe enzyme deficiency or missing.The mortality rate is as high as 50%,and the survivors are often suffered from severe neurological sequelae.The late onset patients can experience repeatedly hyperammonemia episode in different degree,often complicated with nervous system damage,seriously affecting human health and quality of life.However,due to the rarity of UCD and the atypical clinical symptoms,it is difficult to identify,diagnose or treat UCD clinically,as a result,the mortality and disability rates remain high.Therefore,early identification,early diagnosis and early intervention of UCD patients are of great significance to retain the lives of patients and improve the prognosis of patients.Induced pluripotent stem cells(iPSC)is acquired by reprogramming somatic cells with pluripotent factors and generating features and functions of stem cells.Owing to the wide cell source,mature induction and culture techniques,long-term culture ability,carring the same mutation genes with patients,iPSC is an ideal kind of cells to establish disease cell modles.After induce iPSC to target cells,it can be used to establish disease model,compare therapeutic methods,screen drugs and study toxicology,and be used in regenerative medicine field,which has promising prospect.Based on the above research background,this thesis focuses on the summary of clinical data of UCD patients and the construction of IPSCs and hepatic organoids derived from Peripheral Blood Mononuclear cells(PBMC),in an attempt to provide evidence-based medical evidence for the diagnosis and treatment of UCD,and establish in vitro cell models of OTCD.Objectives1.Summarized and analyzed the clinical characteristics,disease course,laboratory examination and genetic test results of 7 children with UCD,to sum up disease characteristics,and explore the relationship between genotype and phenotype.2.Established iPS cell lines derived from PBMC in children with OTCD and subsequently differentiate into liver organoid by 3D culture technology,in order to serve as an in vitro disease model for the study of OTCD.Methods1.A total of 16,154 children at high risk of genetic metabolic diseases who were presented in Qilu Children’s Hospital of Shandong University from October 2016 to December 2020 were screened by blood and urine tandem mass spectrometry.Meanwhile,plasma ammonia test and genetic examination were performed on children with clinical symptoms of encephalopathy such as disturbance of consciousness and convulsion.A total of 7 children with UCD were diagnosed.The results of clinical phenotype,laboratory examination and gene test were analyzed.2.PBMC of OTCD children with different genetic variants was isolated,and reprogrammed by non-integration method.Clones with stem cell morphology were selected for purification and culture.The pluripotent molecular markers of iPSCs were identified by cellular immunofluorescence technique.The expression of endogenous pluripotent genes was detected by qRT-PCR.The expression of molecular markers in the three germ layers was detected by embryoid body self-differentiation assay to verify the multidirectional differentiation potential of iPSCs.The unintegrated reprogramming pattern was verified by PCR to prove that the expression of the foreign gene disappeared.Chromic karyotype identification,qRT-PCR and Sanger sequencing were used to confirm that the constructed iPS was patient specific for OTCD.3.The iPSCs of OTCD children and normal children were both induced to differentiate into definitive endodermal cells(DEC)at the same time,and then cultureed using 3D culture method to differentiate into liver organoid.The expression of specific genes and liver characteristic proteins at different stages were detected by qRT-PCR and immunofluorescence respectively,which proved the successful construction of liver organs.The expression of OTC in liver organoid was detected at both gene and protein levels to preliminarily explore the influence of newly emerged variation on OTC.Results1.Clinical and laboratory data:Among the 7 UCD cases,6 of them belonged to early onset type and 1 with late onset type,all of which showed the clinical process of acute hyperaminemia,combined with multiple organ dysfunction,but neurological symptoms were the prominent manifestations.Coagulation dysfunction precedes liver enzyme abnormality,and the decrease of fibrinogen is significantly correlated with the increase of plasma ammonia.All the patients with early onset UCD were complicated with hypocalcemia,and the serum alanine and proline levels were significantly increased.After receiving blood purification treatment,although the plasma ammonia level was significantly decreased in 2 patients,the clinical symptoms were not significantly improved.2.Genetic results:6 new variations were found in 7 cases,including 5 spot variations and 1 deletion of exon 3-9,all of which were located in the conserved region of genes.The genetic variations that cause early onset cases all result in altered protein structure.By comparing genotypes and clinical phenotypes,it was found that the children with 2 nonsense variations and the deletion of exon 3-9 had more rapid disease progression,or more severe clinical symptoms and poor treatment effect.3.Establishment and identification of iPSCs:After reprogramming,PBMC from children with OTCD showed significant embryonic stem cell morphology.The two OTCD-iPSCs obtained by reprogramming could express pluripotent markers such as TR-60,TR-81,OCT4,SSEA4 and Nanog,as well as endogenous pluripotent genes such as OCT4,SOX2 and Nanog.Both of there two iPSCs can self-differentiate into typical embryomoid structures with expressing specific markers of endoderm,mesoderm and ectoderm.The constructed iPSCs had the same chromosome karyotype as the children’s peripheral blood samples,and retained the original gene variation of cell origin and unintegrated plasmid genes.4.Establishment and identification of liver organoid:The cell bright field maps at different stages showed that iPS cells underwent different stages and finally presented hepatocyte morphology.With the differentiation of iPSCs toward hepatocytes,the expression of markers in the formalized endoderm decreased at both gene and protein levels,while the expression of characteristic markers of hepatocytes,such as ALB,CYP3A4,AFP,were all gradually increased.The PBMC-derived liver organoid of the children with exon deletion 3-9 did not express the cell-derived exon deletion,and the expression of OTC protein was also significantly decreased.Conclusion1.Coagulopathy may be an earlier manifestation of liver injury in patients with UCD,and dynamic monitoring of serum calcium levels in patients with early onset of UCD is needed.Serum alanine and proline levels may be of auxiliary diagnostic value in early onset proximal UCD patients.2.Blood purification therapy can effectively reduce plasma ammonia concentration,however,the improvement of clinical symptoms may not be proportional to the decrease of plasma ammonia.Its clinical phenotype and prognosis still depend on the effect of gene variation on the structure and/or function of the enzyme.Variations occurring in conserved gene regions,enzyme reactivity domains,severely affecting protein structure,as well as large fragment deletions and meaningless variations may have more pronounced effects on enzymes,leading to more severe clinical phenotypes.3.Two PBMC-derived OTCD patient-specific iPS cell lines were successfully established by non-integrated reprogramming technique.The first liver organoid derived from PBMC for this disease was successfully established by inducing it to liver cells,which can be used as a potential liver organ model for OTCD for related studies.