Clinical And Mutational Analysis Based On A Comprehensive Database Of Duchenne Muscular Dystrophy And Dystrophin Gene Modification By CRISPR/Cas9 System

Background and objectives The development of clinical trials for Duchenne muscular dystrophy(DMD)in China faces many challenges due to limited information about epidemiological data,natural history and clinical management.To provide these detailed data,we developed a comprehensive database based on registered DMD patients from South China and analysed their clinical and mutational characteristics.Methods 1.The database included DMD registrants confirmed by clinical presentation,family history,genetic detection,prognostic outcome,and/or muscle biopsy.Clinical data were collected by a registry form.2.Large deletion/duplication mutations of dystrophin gene were detected by multiplex ligation-dependent probe amplification(MLPA).3.Small mutations were detected by Sanger sequencing.Results1.Currently,141 DMD patients from 137 families in South China have been registered,and 91.5% of them were under 10 years old.In mutational detection,large deletions were the most frequent type(57.7%),followed by nonsense mutations(15.3%),large duplications(10.2%),small insertion mutations(7.3%),small deletion mutations(5.8%)and splice site mutations(2.9%).Forty-four small mutations were identified in the probands with negative MLPA,and twenty-four of them were novel mutations that were not reported in the Leiden Open Variation Database.2.Clinical analysis revealed that most patients reported initial symptoms between 1 and 3 years of age,but the diagnostic age was more frequently between 6 and 8 years.85.1% of patients were ambulatory.Baseline cardiac assessments at diagnosis were conducted in 42.6% and 34.0% of patients by echocardiograms and electrocardiograms,respectively.Only 23.4% of registrants performed baseline respiratory assessments.A small number of patients(21.3%)were treated with glucocorticoids.3.15.3% of patients were eligible for stop codon read-through therapy,and 47.4% of patients would potentially benefit from exon skipping.The top five exon skips applicable to the largest group of registrants were skipping of exon 51(14.6% of total mutations),53(11.8%),45(7.3%),55(4.4%),and 44(3.6%).Conclusion 1.Our database provided information on the natural history,diagnosis and management status of DMD in South China,as well as potential molecular therapies suitable for these patients.This comprehensive database will promote future experimental therapies in China.2.Large deletions were the most prevalent mutation,followed by nonsense mutations,large duplications,small insertions,small deletions,and splice site mutations.3.Twenty-four novel mutations in dystrophy gene were identified in the study.4.An analysis of the natural history of DMD in South China showed an obvious diagnostic delay.The rate of baseline cardiac and respiratory assessment at diagnosis was also far from perfect.The use of glucocorticoids in China was relatively low.5.Nonsense stop codon read-through therapy could be applied to 15.3% of DMD patients.About half DMD patients would potentially benefit from exon skipping.The top five choices for single-exon skipping treatments were the skipping of exon 51,exon 53,exon 45,exon 55,and exon 44.Background and objectives The natural history of Duchenne muscular dystrophy(DMD),especially the motor function change,is essential when planning clinical trials.Most of clinical trails have used the progress of motor function to evaluate the curative effect of treatment.Assessment for motor function of DMD patients with the standard measures used in clinical trails is very valuable for the design and development of clinical trails.Our previous study showed obvious diagnostic delay and low rate of glucocorticoid treatment in DMD patients from South China,which may result in specific characteristics of motor function chage.By now,motor function of DMD in China has no been systematically studied.Moreover,no study has been performed to evaluate motor function with standard measures in Chinese DMD patients.Based on our comprehensive database,we conducted a prospective observational DMD cohort and assessed the motor function with the recommended standard measures to analyze the detailed information on motor function change in Chinese DMD patientsMethods 1.Included patients were from our DMD database and should fill with the inclusion criteria for further motor function assessment.2.Baseline motor function of patients was tested by the standard measures,including 6-minute walk test(6MWT),North Star Ambulatory Assessment(NSAA),10-meter timed walk/run test and Gowers' test.3.Reevaluation of motor function was performed by standard measures after 12 months(±2 months).4.A 1:1 matched pair study was conducted to detect the difference of motor function between the two groups of DMD patients carried large deletion/duplication mutations and small mutations.Results1.In the baseline assessment,the 6MWT distances and NSAA scores decreased with age after 7 years old,while time of 10-meter timed test increased with age after 7 years.Gowers' time increased with age.In the patients over 7 years old,the regression analysis showed that 6MWT distances decreased on the average of 30.4 meters per year(P<0.05),NSAA scores decreased on the average of 2.7 scores per year(P<0.05)and time of 10-meter timed test increased on the average of 1.0 seconds per year(P<0.05).Gowers' time increased on the average of 1.1 seconds per year(P<0.05).2.The results of 12-month followed up showed the amplitude of 6MWT change over 12 months increased on the average of 46.3 meters per year(P<0.05).Compared with no steroid group,6MWT distances in the steroid group increased on the average of 79.5(P<0.05).The amplitude of NSAA change over 12 months increased on the average of 1.7 scores per year(P<0.05).Compared with no steroid group,NSAA scores in the steroid group increased on the average of 3.7(P<0.05).3.All the measures in baseline assessment were highly correlated(P<0.05).4.In the 1:1 matched pair study,no difference of motor function was found between the two groups of DMD patients carried large deletion/duplication mutations and small mutations.Conclusion 1.The motor function change of DMD patients from South China in our study has obvious characteristics.Most of patients achieved their best motor function at 7 years old.After the age of 7,both the 6MWT distances and NSAA decreased with age progressively,while time of 10-meter timed test and Gowers' time increased with age.The motor function deteriorated with age significantly after 7 years old.2.The amplitude of 6MWT and NSAA change over 12 months increased with age.3.DMD patients benefitted from glucocorticoids,which improved both 6MWT distances and NSAA scores.4.No difference was found in motor function between DMD patients with different mutations.Background and objectives With the development of basic researches and clinical trails on therapy,some genetic-based methods have been found to treat Duchenne muscular dystrophy(DMD)patients with large deletion mutations and nonsense mutations.However,appropriate therapeutic approach for repairing duplication mutation is limited.Duplication mutation is predicted to produce full-length dystrophin protein after modification.This advantage not only brings hope for complete treatment to patients with duplication mutations,but also helps to further explore the fully efficiency and application value of specific therapeutic methods in DMD therapy.As a new genome-editing tool,CRISPR/Cas9 system is capable to make precise genome modifications,which may be used in the edting of duplication mutations.In this study,we used the CRISPR/Cas9 system to correct a duplication mutation based on patient-derived myoblasts.The aim of this study was to explore the potential efficiency and apply of CRISPR/Cas9 system in correcting duplication mutations.Methods 1.Muscle tissues from a DMD patient with duplication of dystrophin were obtained and were cultured for primary cells.Immunohistochemistry stain was performed to detect dystrophin level in the serial frozen sections.2.Sanger sequencing was used to detect breakpoint of duplication mutation in the DMD patient.3.Myoblasts were purified with an immuno-magnetic sorting system using CD56 microbeads.Purified myoblasts were determined by immunofluorescence staining for desmin.After differentiation for 7 days,myoblasts were further confirmed by immunofluorescence staining for MHC.4.Designed sgRNA was cloned to Px458 vector.Plasmid was transformed into HEK293 T.T7 endonuclease was used to test functional validation of sg RNA.5.Myoblasts were transducted by lentivirus with a lenti-CAS9-sg RNA vector and differentiated for 7 days.6.Real-time PCR detected the copy number variation of exon 6 and exon 25 in the patient's myotubes before and after genetic editing.7.Off-Target Analysis was conducted by Sanger sequencing for top 10 off-target hits.8.Western blot was performed to detect the present of full-length dystrophin protein in the patient's myotubes before and after genetic editing.9.Immunofluorescence staining for dystrophin and syntrophin was used to detect recovery of dystrophin-glycoprotein complex(DGC).Results 1.The patient carried duplications of exon 18-25.No insertion sequence was found at the breakpoint junction.Immunohistochemistry stain showed completely deficiency of dystrophin protein.2.Immunofluorescence staining showed more than 85% of the selected CD56(+)cells stained with anti-desmin antibodies.After differentiation for 7 days,the CD56(+)cells changed into multinuclear fusiform myotubes and stained with anti-MHC antibodies.3.In functional validation test of sgRNA,the control group only showed one band from PCR product.The sg RNA group was digested by T7 endonuclease into two products and showed another two bands with the same size to the target cleavage bands.4.The result of real-time PCR showed copy number of exon 6 in patient's myotubes was 0.841±0.029 compared with that of the health control.After editing,copy number of exon 6 in patient's modified myotubes was 0.889±0.023 compared with that of the health control.The difference was not statistically significant(P=0.084).Copy number of exon 25 in patient's myotubes was 2.015±0.079 compared with that of the health control.After editing,copy number of exon 25 in patient's modified myotubes was 1.308±0.083 compared with that of the health control.The difference was statistically significant(P<0.001).5.Off-target analysis revealed no abnormal editing was found in the predicted top 10 off-target sites.6.Western blot showed patient's myotubes expressed no dystrophin protein before editing.After editing,the patient's myotubes expressed full-length dystrophin protein with a quantity about 6.12% of health control.7.Immunofluorescence staining showed recovery of both dystrophin and syntrophin.Conclusion 1.CRISPR/Cas9 system was capable to remove the whole duplicated DNA sequence in DMD multiple-exon duplication.2.The modification of duplicated DNA sequence by CRISPR/Cas9 system restored the expression of full-length dystrophin.This genome-editing method could be applied to future genetic therapy for DMD patients with duplication mutations.3.CRISPR/Cas9 system had specific advantages in editing DMD duplication mutations for that only one sg RNA was able to remove the whole duplicated region.