The Molecular Study Of Hereditary Spastic Paraplegia Caused By ERLIN2 Mutation Based On The HiPSC Model

Background and ObjectiveHereditary neuromuscular disease（HNM）are a group of neuromuscular diseases characterized by muscle atrophy and weakness,with significant genetic and phenotypic heterogeneity.The complexity of clinical and genetic classification brings great challenges to the diagnosis and treatment of the disease.Hereditary Spastic Paraplegia（HSP）is a type of hereditary neuromuscular disease,which is clinically characterized by slowly developing muscle atrophy and retarded spastic paraplegia of the lower limbs.Up to now,78 subtypes of HSP have been identified,including 80 pathogenic genes,with significant diversity in inheritance patterns.New pathogenic genes and new pathogenic variants are still being discovered.The study of the molecular mechanism of HSP related genes not only contributes to a deeper understanding of the disease process,but also provides a theoretical basis for the exploration of the treatment of HSP.We have previously conducted a systematic collection and clinical genetic analysis of HNM patients in the region,established a HNM cohort of 69 patients,and completed the molecular diagnosis of 31 patients.A heterozygous missense mutation in ERLIN2 gene was identified in a clinically diagnosed HSP pedigree with autosomal dominant inheritance by sequential genetic testing,which had not been reported in the literature and databases before.Previous studies have found that biallelic loss-of-function mutations in the ERLIN2 gene can lead to autosomal recessive HSP 18,but the specific pathogenesis is not clear,and it is difficult to effectively guide the diagnosis,treatment,and prevention of the disease.More importantly,several problems still remain to be determined.Is there an autosomal dominant inheritance mode of HSP 18?Does heterozygous missense mutation of ERLIN2 cause HSP18 through gain of function（GOF）mechanism?Is there a common molecular mechanism between the two modes of inheritance?The traditional view is that HSP is a typical neurodegenerative disease,which usually begins in adolescence and even adulthood,and gradually worsens with age.In recent years,a growing number of studies have suggested that neurodegenerative diseases have a developmental genetic basis,which has significant defects in the stage of neurodevelopment.The disorder of neural differentiation leads to the reduction of the number of neurons and shows typical clinical symptoms due to neurological decompensation in the onset stage.In recent years,the rapid development of induced pluripotent stem cells（iPSC）and gene editing technology can well simulate the occurrence and development of genetic diseases,which is a good in vitro model for studying the pathogenesis of genetic diseases.It plays an irreplaceable and important role in the analysis of the pathogenesis of genetic diseases.Therefore,using patient-derived iPSCs and ERLIN2 biallelic knockout iPSC models based on CRISPR/Cas9 technology,this study aims to investigate:1.Whether the ERLIN2gene mutation has an autosomal dominant pathogenic mode in HSP18;2.What is the pathogenic mechanism of heterozygous missense mutation of ERLIN2 gene;（3）whether the different mutation types and inheritance patterns of ERLIN2 gene have a common molecular mechanism.Materials and Methods1.Clinical data were collected,and mutation analysis was carried out.With the informed consent of the family members and the approval of the Ethics Committee of Army Medical University,the relevant clinical data of the family members were collected,and their peripheral blood samples were drawn for sequential genetic testing.The whole exome sequencing data were subjected to variant screening,polymorphism database comparison,Sanger sequencing verification,co-segregation analysis,and mutation harmness prediction to identify the candidate pathogenic variants in the family.2.Mechanism of HSP caused by a heterozygous missense mutation of ERLIN2.2.1 Construction of patient-derived iPSC model.The peripheral blood of the patients and their family members was collected,and the Erythroid Progenitor cells were isolated and cultured.The Erythroid Progenitor Reprogramming kit was used to induce reprogramming to obtain iPSC lines with the same genetic background.2.2 Pathogenic mechanisms.iPSCs derived from the patient were set as the case group,and iPSCs derived from normal family members were set as the control group.iPSCs were induced to differentiate into motor neurons,and detected the differentiation efficiency.iPSCs were induced to differentiate into neural stem cells,and protein-protein interactions were analyzed by IP-mass spectrometry.Cell proliferation and apoptosis were detected by flow cytometry.Western Blot was used to detect the expression of apoptosis-related proteins.Laser confocal microscopy was used to image the intracellular calcium flow and analyze the changes of intracellular calcium level.The transcriptome of neural stem cells in the case group and the control group was sequenced,and the differentially expressed genes were analyzed by GO,KEGG and GSEA bioinformatics to explore the changes of gene expression and enriched signaling pathways in NSCs in the case group.3.Mechanism of biallelic deletion of ERLIN2 in HSP.3.1 Construction of ERLIN2 biallelic deletion stem cell model.The gRNA sequence with high specificity and knockout efficiency was designed by software.The p Sp Cas9（BB）-2A-EGFP vector was used to construct the plasmid,and the biallelic knockout monoclonal cells were selected and identified after nuclear perforation transfection.A model of biallelic deletion of the ERLIN2 gene was obtained by using SBU cells derived from normal human,which were identified by sequencing analysis,m RNA expression detection and protein content determination.3.2 Pathogenic mechanisms.The ERLIN2 KO hiPSC line was induced to differentiate into neural stem cells,and the apoptosis level and cell cycle of neural stem cells were detected.The expression of apoptosis-related proteins and cell cycle proteins was detected by Western Blot.Laser confocal microscopy was used to image the intracellular calcium flow and analyze the changes in intracellular calcium level.The difference of mitochondrial membrane potential was detected and analyzed.ResultsThe clinical data of the family were integrated and the sequential genetic testing techniques,including dynamic mutation analysis,neuromuscular disease gene panel,MLPA and whole exome sequencing,were used.A heterozygous missense mutation,ERLIN2（c.212T>C,p.Val71ala）had not been reported previously.Based on the results of the mutation harm prediction tool and ACMG guidelines,the mutation was considered to be a suspected pathogenic mutation,which may cause HSP through an autosomal dominant inheritance pattern.Patient-derived iPSCs were obtained by culturing erythroid progenitor cells from the patient’s peripheral blood and reprogramming.The patient-derived iPSCs carried patient-specific genetic information,grew actively,expressed pluripotency proteins,and had multi-lineage differentiation potential.Based on a patient-derived iPSC model,we explore the pathogenic mechanism of the ERLIN2 p.Val71Ala mutation.The results suggested that the efficiency of iPSC differentiation into motor neurons was significantly reduced,and the proliferation of nerve stem was blocked,and the apoptosis was significantly increased.ERLIN2 p.Val71ala mutation enhances its function by recruiting E3 ubiquitin ligase RNF213 to promote the degradation of IP3R1,block the outflow of Ca²⁺from the endoplasmic reticulum,disrupt intracellular calcium homeostasis,and lead to pathological endoplasmic reticulum stress,thereby affecting the apoptosis of neural stem cells.Endoplasmic reticulum stress inhibitor TUDCA can partially rescue the apoptotic phenotype of NSCs.KEGG analysis of transcriptome sequencing and WB results suggested that the disruption of intracellular calcium homeostasis caused by ERLIN2 mutation inhibited the activation of the MAPK signaling pathway,thereby inhibiting cell proliferation.The ERLIN2 gene biallelic deletion（ERLIN2-KO）iPSC model constructed by CRISPR/Cas 9 technology,which achieved homozygous knockout of the gene,no ERLIN2protein expression was detected in the cells,and the stem cell characteristics of the cell line were maintained.The ERLIN2-KO iPSC model was induced to differentiate into NSCs,and cell apoptosis and cycle detection showed that the level of cell apoptosis was significantly increased,and the cell cycle was arrested in G2/M phase.Loss of ERLIN2 protein leads to the blocking of IP3R1 degradation in NSCs,further increasing Ca²⁺transport to mitochondria and mediating apoptosis by reducing mitochondrial membrane potential.At the same time,ERLIN2 deficiency weakens the stability of cyclin B1,leads to cell cycle arrest,and reduces the proliferation ability of NSCs.ConclusionERLIN2 is an important protein in cells to maintain calcium homeostasis and the ERAD pathway.In addition to autosomal recessive inheritance,heterozygous missense mutations of ERLIN2 can also lead to HSP through autosomal dominant inheritance mode.Heterozygous missense mutation of ERLIN2 can recruit E3 ubiquitin ligase RNF213,enhance its degradation of IP3R1,block calcium efflux from ER,and cause pathological ER stress response.Biallelic deletion of ERLIN2 causes loss of its protein function and releases its degradation of IP3R1 protein,leading to increased endoplasmic reticulum Ca²⁺efflux and mitochondria-mediated apoptosis.There are significant differences in the molecular mechanisms of the two mutation patterns,but both cause disease by disrupting intracellular calcium homeostasis,which provides a basis for explaining the phenotypic and genetic heterogeneity of HSP and contributes to the exploration of therapeutic strategies for HSP.