Analysis Of Patients With CblC Type Methylmalonic Acidemia And The Establishemt Of IPSCs And Mouse Model

Part 1 Clinical data analysis of patients with cbl C type methylmalonic acidemiaObjective: To analyze the clinical manifestations,mass spectrometry results,genotype,treatment effect and prognosis of patients with combined methylmalonic acidemia,figure out the correlation between genotypes and phenotypes,so as to help enhancing the understanding of this disease for clinicians.Methods: Retrospective analysis a data of 566 patients with combined methylmalonic acidemia caused by mutation of MMACHC gene in our hospital from August 2009 to January 2019,including age of onset,clinical manifestations,blood tandem mass spectrometry combined with urinary gas chromatography results,brain MRI,gene detection results,treatment and prognosis.Analysis the genotype-phenotype correlation of cbl C type MMA.Results: Of the 566 patients,195(34.5%)were from newborn screening,52(26.7%)of which were clincal patients;30(8.1%)of 371 unscreened patients were diagnosed after their siblings' onsets and the remaining 341(91.9%)were clinical patients.The median age of onset was 3 months(1 day to 26 years),340(86.5%)of early-onset patients were more likely to present with feeding difficulty,mental/motor retardation,lethargy,convulsions,and vomiting;while 53(13.5%)lateonset patients were mainly presenting with decreased academic performance,memory loss,poor expressionand and decreased exercise capacity.All patients were vitamin B12-responsive.After treatment,the blood level of propionyl carnitine,the ratio of propionylcarnitine to acetylcarnitine,the urinary level of methylmalonic acid,methylcitrate and homocysteine decreased significantly(P<0.05).Brain MRI showed that ventricles enlargement,widening of the extra cerebral space and dysplasia of the corpus callosum are common manifestations.The MMACHC gene detection results showed that the hot spot mutation was c.609G>A(p.W203X)(32.95%),followed by c.658-660 del AAG(p.K220del)(13.07%),c.80A>G(p.Q27R)(7.69%),c.567 dup T(p.I190Yfs*13)(6.27%),c.482G>A(p.R161Q)(5.3%)and c.394C>T(p.132X)(3.89%).Patients with c.609G>A(p.W203X),c.658-660 del AAG(p.K220del)and c.567 dup T(p.I190Yfs*13)often had an early onset within 1 year old;patients with c.482G>A(p.R161Q)were more likely to be late-onset.494 patients were followed up,150(83.3%)patients who diagnosed from newborn screening were normal,26(14.4%)had development delay,and 4 died(2.2%);91(29.0%)of the unscreened patients developed well while 186 patients(59.2%)were underdeveloped and 37 died(11.8%).Conclusions: Patients with cbl C methylmalonic acidemia are more likely to have an onset(80%)within 1 year of age.The most common manifestations are feeding difficulty,sports/intellectual disorder,convulsions and lethargy;20% patients are lateonset with decreased academic performance,cognitive impairmentand.Ventricular enlargement,widening of extracerebral space and dysplasia of the corpus callosum are common MRI findings.The MMACHC gene c.609G>A is a hot spot mutation in China.c.609G>A(p.W203X),c.658-660 del AAG(p.K220del)and c.567 dup T(p.I190Yfs*13)are associated with early onset while c.482G>A(p.R161Q)is associated with late onset.Newborn screening is conducive to early diagnosis and long-term prognosis of cbl C type methylmalonic acidemia.Part2 Differentiation of cbl C type methylmalonic acidemia hi PSCs into neuronsObejective: To establish methylmalonic acidemia hi PSCs and differentiate them into neurons,which may shed light on discovery of early biomarkers and pathways dysregulated in MMA,as well as provide a basis for novel therapeutic strategies to treat MMA.Methods: Construction of disease-specific hi PSCs by reprogramming peripheral blood mononuclear cells with homozygous mutants of MMACHC c.609G>A,use generation sequencing,alkaline phosphatase staining,RT-PCR,immunofluorescence and teratogenesis assay to identify the pluripotency of hi PSCs,karyotype analysis to detect its safety;(2)Use CRISPR/Cas9 gene editing technology to repaire MMACHC W203X/W203 X hi PSCs and carry out correlated tests;(3)Use EB protocols to induce hi PSCs into neural progenitor cells and neurons.Results:(1)This hi PSCs was MMACHC c.609G>A homozygous;positive in cloned alkaline phosphatase staining test;expressed pluripotency genes SOX2,REX1,OCT4,NANOG,LIN28;had a narmal male karyotype(46,XY);positive stem cellspecific antigens were;showed a tumorigenicity and three germ layer differentiation ability.(2)CRISPR/Cas9-mediated targeted gene editing corrected the hi PSCs to heterozygous mutation with the genotype of MMACHC +/W203X;ins T.(3)Both MMACHC W203X/W203 X hi PSCs and MMACHC +/W203X;ins Thi PSCs can be induced into neural precursor cells and neurons;MMACHC W203X/W203 X hi PSCs showed a decreased differentiation efficiency and prolonged neuronal maturation in neural induction systems.Conclusion: We successfully established cbl C type MMA disease-specific hi PSCs,namely MMACHC W203X/W203 X hi PSCs and correct the mutation by CRISPR/Cas9(MMACHC +/W203X;ins T).MMACHC W203X/W203 X hi PSCs suffered a lower neural induction differentiation efficiency and prolonged neuronal maturation time.Part 3 Establishment a systemic inducible mouse model of cbl C type methylmalonic acidemiaOBJECTIVE: To Establish a systemic inducible cbl C-type MMA mouse model which provide a basis for exploring the pathogenesis of this disease.METHODS: Mmachc gene conditional knockout mice(Mmachcflox/+ mice)were constructed using CRISPR/Cas9 technology.F1 generation mice were crossed with UBC-Cre ERT2 mice.The 40~50-day-old Mmachcflox/flox;UBC-Cre ERT2 male mice(experimental group)and Mmachcflox/+;UBC-Cre ERT2 male mice(control group)were intraperitoneally injected with Tamoxifen for 5 days to induce Mmachc gene whole-body knockout.The body weight changes and manifestations were recorded.The Mmachc gene knockout efficiency was detected by PCR.The expression of Mmachc was detected by RT-PCR.The changes of C3 and C3/C2 in tail vein blood of mice before and after Tamoxifen induction were detected by tandem mass spectrometry.RESULTS: Intraperitoneal injection of Tamoxifen for 5 days induced a whole-body knockout of Mmachc gene in Mmachcflox/flox;UBC-Cre ERT2 mice.Two 40-day-old experimental mice showed decreased body weight and activity with breath difficulty which eventually died within 8 days.Three 50-day-old experimental mice and the control group showed no symptoms.RT-PCR showed a decreased Mmachc expression level in brain,heart,liver,spleen,lung and kidney of Mmachcflox/flox;UBC-Cre ERT2 mice after Tamoxifen treatment.The blood levels of C3 and C3/C2 in Mmachcflox/flox;UBC-Cre ERT2 mice were increased after Tamoxifen induction(P<0.05).Conclusion: We established a systemic inducible cbl C-type MMA mouse model.There may be a correlation of induction time-onset time in our mouse model.