The Genetic Testing Of Muscular Dystrophy And Study Of The Diagnosis Strategy Of Difficult Cases

Part 1 Study on gene detection and mutation spectrum of muscular dystrophy in Guangxi regionObjective The genetic diagnosis process of muscular dystrophy(MD)was applied to the diagnosis and analysis of suspected patients with MD in Guangxi region.The gene mutation spectrum of Duehenne/Becker muscular dystrophy(DMD/BMD)in Guangxi area was summarized.The relationship between genotype and phenotype was analyzed,and accurate genetic counseling and prenatal diagnosis were provided.It had laid the foundation for exploring new treatment scheme and deepened the understanding of MD.Methods 1.The clinical data,laboratory test results and blood samples of suspected MD patients and family members were collected.2.Multiplex ligation dependent probe amplification(MLPA)was used to detect the DMD gene in suspected MD patients.Parents,sibs and maternal family members verification of patients with positive results was carried out.The mutation hotspots,breakpoint analysis,reading frame detection and exon skipping therapy suitability analysis were analyzed and compared with the detection data from other regions.3.The hereditary myopathy Panel based next generation sequencing(P-NGS) was carried out on the gene detection of patients with negative MLPA results.The positive loci detected by Panel sequencing were annotated,analyzed and filtered and further verified by Sanger sequencing.Their families were analyzed as well.4.The patients whose Panel results were negative that would be further detected by other methods such as whole-exome sequencing(WES),wholegenome sequencing(WGS),or massively parallel sequencing of RNA(RNA-seq).Results 1.A total of 311 patients with suspected MD were included for genetic testing,of which 294(94.5%)patients were confirmed.Among them,274(88.1%)cases were DMD/BMD,20(6.4%)cases were other types of myopathy(including 8 cases of Limb-girdle muscular dystrophy,3 cases of Bethlem myopathy,3 cases of Glycogen storage disease,2 cases of Spinal muscular atrophy,2 cases of Emery-Dreifuss muscular dystrophy,and 1 case of Nemaline myopathy),and 17(5.5%)cases failed to be diagnosed at the genetic level.2.(1)A total of 201 patients with large fragment mutations in the DMD gene were detected by MLPA,of which 4 were female DMD patients,with a positive diagnosis rate of 64.6%(201/311).There were 100 different exon mutation modes,of which 137 patients had multiple exon deletions(the largest missing fragment among them was exon 1-60),while 31 patients had a single exon deletion,and the rest 33 patients had a large duplication.(2)Analysis of mutation hot spots: The hot spots with large fragment deletions are mainly concentrated in exons 45-54,with exon 49 being the most frequently involved.The hot spots with large fragment duplications are mainly concentrated in exons 2-13 and 46-50,with exon 9 are the most frequently involved.(3)Analysis of the breakpoints: The hot spots of the large fragment deletion breakpoint were introns 44-52,with most frequently involving intron 44.The hot spots of the initial breakpoint were introns 44-50,and most frequently involving intron 44.The hot zones of the termination breakpoints were introns 48-55,and intron 50 was most frequently involved one.There were no obvious hot spots at the breakpoints of large fragment duplication,and the frequency of introns 1,2,7,44,45,and 62 was slightly higher,with introns 1 and 2 were the most.The initial breakpoints were more often involved in introns 1,2 and 45,with intron 1 were the most,and the termination breakpoints usually involved introns 2,9,13 and 62.(4)Detection of reading frame: Among 201 cases of large fragment deletion and duplication mutations,32 cases(15.9%)were inframe mutations,164(81.6%)were out-of-frame mutations,and 4 case(2.5%)could not be confirmed as in-frame or out-of-frame mutation.Seven patients were too young to be classified for the clinical phenotype as DMD or BMD.Among 193 patients with large deletions and duplication mutations confirmed by clinical phenotype,173 patients(89.6%)with DMD/BMD conformed to the reading frame rule.(5)Applicability analysis of exon skipping therapy: Twenty-eight patients with DMD may be suitable for exon 51 skipping therapy,24 patients may be suitable for exon 53 skipping therapy,and 4 patients with exon 52 deletion were both suitable for exon 51 and 53 skipping therapy.A total of 48 patients may be suitable for exon 51 or 53 skipping therapy,accounting for 28.6% of all exon mutation patients and 17.5% of all DMD gene mutation patients.3.Detection of point mutation: The hereditary myopathy Panel based NGS was performed on 110 cases with negative MLPA results.71 cases(64.5%)were diagnosed with point mutations in the DMD gene,of which 33 cases(46.5%)were nonsense mutations,and 5 cases(7.0%)were missense mutations,18 cases(25.4%)were splicing mutations,and 15 cases(21.1%)were frameshift mutations.Among them,40 cases(56.3%)had unreported new mutation sites,which were predicted to be pathogenic mutation sites by bioinformatics software.4.Detection of carriers: The test for carriers were carried out on 322 female relatives in 204 DMD/BMD families,of which 180(55.9%)female carriers were detected in 138(67.6%)families,and the genetic mutation of 136 patients(66.7%)were inherited from their mother.In another two cases,their mother’s blood samples were negative for genetic testing,but they both gave birth to two consecutive offspring with the same genetic mutation,so they were considered to be suspected gonadal chimera carriers.Conclusions 1.MLPA detection of DMD gene combined with hereditary myopathy Panel based NGS is currently the best genetic testing strategy for the diagnosis of suspected MD patients.2.Patients with suspected MD in Guangxi region are mainly DMD/BMD.Large deletion/duplication mutation is the most common DMD mutation type.The hot spots of large fragments mutations and breakpoints of DMD gene.There are basically consistent with other studies.89.6% of DMD/BMD patients in Guangxi conformed to the reading frame rules.The clinical diagnosis and prognosis prediction should be based on the reading frame destruction and patient phenotypes.3.The point mutations of DMD gene in Guangxi region are mainly nonsense mutations,which is consistent with other studies.And 40 cases of point mutations are new mutation sites,including nonsense mutation,frameshift mutation and splicing mutation,which enriched the mutation spectrum of DMD gene.4.In this study,48 patients of DMD with large fragment deletions and 33 nonsense mutations may be suitable for existing gene therapy.5.66.7% of DMD patients in Guangxi have genetic mutations inherited from their mothers,which conforms to the incidence rules of DMD gene mutations.Clinicians and geneticists should consider the possibility of gonad chimerism when conducting molecular tests to reduce the risk that patients with the same genetic mutation will reappear in their family.Part 2 Pedigree study of rare Xp21 contiguous gene deletion syndrome caused by the complex genome rearrangementsObjective Based on the clinical manifestations of typical DMD and Adrenal hypoplasia congenita(AHC)in two brothers in a family,as well as the fact that genetic tests were negative.The WGS reanalysis technique was used to identify new rearrangement events in the Xp21.2-3 region caused by complex breakage and inversion,providing a gene-level diagnosis for a family with a rare Xp21 contiguous gene deletion syndrome.Methods 1.The clinical data,laboratory test results and blood samples were collected.2.Skeletal muscle MRI,HE staining and dystrophin immunohistochemical staining of muscle biopsy,serum muscle-specific mi RNAs,urinary organic acid gas chromatography-mass(GC-MS),chromosomal microarray analysis(CMA),chromosome karyotype analysis of peripheral blood,MLPA of DMD genes,hereditary myopathy Panel based NGS,high-precision clinical exome sequencing and two whole-genome sequencing(WGS)were performed.During the second WGS library construction,the size of the insert was kept at about 350 bp.3.In view of the suspicious positive breakpoints selected by the second WGS screening,the changes in gene structure were analyzed,primers were designed to be verified by PCR and Sanger sequencing,and the effect on gene expression was explained.Meanwhile,family verification and co-separation analysis were performed.Results 1.The clinical manifestations of 2 male children were progressive muscle weakness,mild mental retardation,positive Gowers sign,skin pigmentation,and mild salt loss.Laboratory results showed low sodium,decreased plasma cortisol,increased ACTH,increased CK,CK-MB,ALT and AST,and normal blood glucose and triglycerides.The Gesell intelligence test scale of proband suggested mental retardation.The myocardial enzymes of their mother,eldest sister,and second sister were mildly to moderately elevated,and the proband’s father had normal results.2.The lower extremity skeletal muscle MRI of the two patients indicated that the fat infiltration of the thighs mainly affected the gluteus maximus and adductor magnus,and the fat infiltration of the calves mainly involved the gastrocnemius.The patients’ mother had slight fatty infiltration of gracilis and sartorius,and the patients’ eldest sister did not have fatty infiltration and edema in her lower limb skeletal muscle.3.The HE staining of gastrocnemius biopsy in the two patients showed that the muscle fibers had obvious muscular dystrophy changes.The immunohistochemical staining of Dystrophin N terminal,R terminal,and C terminal antibodies revealed almost complete or complete deletion of Dystrophin protein in the sarcolemmal.4.The relative expression levels of serum miR-499 in the two patients were 10.46 and 56,respectively,both higher than the cut-off value of 4.715 in DMD patients.The probability value of CK combined with mi R-499 to predict the mother of the proband as a DMD carrier was 0.949,greater than the cut-off value of 0.594.5.The results of urine organic acid GC-MS,CMA,chromosome karyotype of the peripheral blood,MLPA of DMD gene,hereditary myopathy Panel based NGS,high-precision clinical exome sequencing,and the first WGS in the two patients were all negative.6.The second WGS results showed that there were 3 breakpoints on the proband’s chromosome Xp21,which were located in the intergenic region with no specific gene,approximately 1.6 kb upstream of NR0B1 gene and the intron 43 of DMD gene,respectively.These breakpoints led to gene rearrangements in the Xp21.2-3 region,which destroyed the structure of DMD and NR0B1 genes,but the complete GK gene was retained.7.Based on the wild-type sequence and the upstream and downstream sequences of the three breakpoints,primers were designed to perform PCR amplification on the proband and its family members.Amplification of three rearrangements sequence was found in the samples of the proband and his younger brother,and no amplification of the wild-type sequence.In the samples of their two sisters and mother,amplifications of the three rearrangements and the wild-type sequence were found.There was only amplification of the wild-type sequence in the samples of the proband’s father.These results were verified by Sanger sequencing.Conclusions 1.The complex genome rearrangements(CGRs)event on Xp21 chromosome resulted in phenotypes of DMD and AHC in two patients who are hemizygous patients with this CGRs event.Both sisters and mother are heterozygous carriers,and the father is a wild type without mutation,following the X-linked recessive inheritance model,which is in line with the family co-separation.2.For the first time,a CGRs event involving only DMD and NR0B1 genes but not GK genes was reported,resulting in a rare Xp21 contiguous gene deletion syndrome with DMD and AHC phenotypes but no GKD phenotypes.3.For the first time,mi R-499 was applied to assisted diagnosis of DMD patients and carriers for the first time,further demonstrating the potential of serum muscle-specific mi RNAs to assist in the identification of DMD patients and carriers.4.For suspected patients with negative results of conventional genetic testing but strong clinical evidence,genetic testing should be reanalyzed to help find the defects of previous tests and improve the diagnostic efficiency.Clinical phenotype is an important indicator for selecting genetic testing methods.When multiple phenotypes exist at the same time,WGS may be a more economical and effective method.Improved insert size of WGS library may help balanced structural variation discovery.