Disease-causing Gene Screening And Reproductive Intervention For Duchenne Muscular Dystrophy

BackgroundDuchenne muscular dystrophy(DMD),a recessive genetic disease with X chromosome,is a congenital defect disease caused by a mutation in the Dystrophin gene,and progressive muscle atrophy in the whole body is caused by a deficiency in anti-muscular atrophy protein.The condition of DMD patients develops rapidly and the prognosis is poor.At present,there is no effective treatment in clinic.Preimplantation genetic diagnosis(PGD)is an effective way to avoid the birth of such children in families with a history of childbearing in DMD patients.Prenatal diagnosis can be performed by villus biopsy at 11-13+6 weeks of gestation or amniotic fluid extraction at 16-24 weeks of gestation to detect whether the fetus carries the pathogenic mutation of Dystrophin gene.If the fetus is detected as a child with DMD,it is necessary to terminate the pregnancy in time.Repeated abortion is easy to cause physical and mental damage to pregnant women and families.With the advancement of IVF technology,PGD technology came into being.PGD is a technology based on in vitro fertilization-embryo transfer(IVF-ET),which uses a series of genetic testing methods to screen out high-quality embryos that do not carry pathogenic genes for transplantation.This technique avoids the pain caused by induced labor during pregnancy to pregnant women and their families.However,due to the high cost of testing,most families with genetic diseases are discouraged.Currently,molecular diagnostic techniques for PGD include Multiplex ligation dependent probe amplification(MLPA),Sanger sequencing,single nucleotide polymorphism(SNP)and short tandem repeats(STR)linkage analysis,etc.Among them,SNP is widely used for linkage analysis in the process of PGD due to its high genome density.However,SNP detection is expensive.Because it is a diallelic polymorphism and provides relatively little genetic information in genetic analysis,it is often necessary to detect more than 60 SNP in order to carry out effective linkage analysis.Therefore,it is of great significance to make pre-embryo transfer diagnosis for families with a history of childbearing DMD,to compare the advantages and disadvantages of STR and SNP in the application of PGD,and to explore more suitable diagnostic methods for PGD technology.Objective1.To conduct the molecular genetics study and identify the pathogenic gene for one Chinese family with Duchenne muscular dystrophy.2.The advantages and disadvantages of STR and SNP in the application of PGD were compared through analysis in order to provide a cheaper,rapid and simple diagnostic method for PGD in genetically deficient pedigrees.MethodsDetailed physical examination and family history investigation of DMD family members were conducted.we used MLPA and Next-generation sequencing(NGS)technology to conduct genetic testing on a suspicious DMD family,and judged the pathogenicity of gene mutation according to ACMG guidelines.In order to avoid reproducing such children again,we implemented the “third generation in vitro fertilization” assisted reproductive technology for this family.The routine ovulation stimulation and intracytoplasmic sperm injection were used to obtain available embryos,and we combined with low-depth whole genome sequencing(CNV-seq),Sanger sequencing,SNP/STR linkage analysis for embryo chromosome screening and gene mutation detection to screen high-quality embryo transfer.After a successful pregnancy,amniotic fluid was extracted during the second trimester for prenatal diagnosis to further verify the results of PGD.ResultsThe proband was an 8-year-old male with clinical manifestations of bilateral lower limb weakness,bilateral gastrocnemius pseudohypertrophy and difficulty in squatting and standing.Laboratory examination showed that serum creatine kinase increased up to 12,000 U/L,and muscle biopsy showed progressive muscular dystrophy.No exon deletion or duplication of Dystrophin was found by MLPA technology.NGS sequencing revealed that the proband was a hemizygous mutation of Dystrophin gene c.829C>T,and her mother was the carrier of the mutation.The mutation was analyzed as a pathogenic mutation in combination with ACMG guidelines,suggesting that the mother had a higher risk of having children with DMD again.Six available embryos(named E1,E2,E3,E4,E5,E6)were obtained by conventional ovulation induction and intracytoplasmic sperm injection.Low-depth whole genome sequencing(CNV-seq),Sanger sequencing and SNP/STR linkage analysis were used to screen embryonic chromosomes and detect the c.829C>T mutation of Dystrophin gene.Two transferable embryos were screened: E5,female embryos and E6,normal male embryos.E6 was preferred for transfer,and embryo implantation failed;then E5 was chosen for transfer,and the pregnancy was successful.Amniotic fluid was extracted at 18 weeks of gestation for prenatal diagnosis to further validate the results of PGD.The results showed that the fetal chromosome was euploid,and the c.829C>T heterozygous mutation of Dystrophin gene,which was consistent with the results of PGD.At the same time,by comparing the results of SNP and STR analysis,it was found that the results of STR was consistent with the SNP linkage analysis.Because STR can provide abundant genetic information,3-5 STRs can carry out effective linkage analysis.Therefore,it has the advantages of low detection cost,short time-consuming and easy operation.Conclusions1.The nonsense mutation of c.829C>T in exon 8 of Dystrophin gene was the cause of DMD patients in this study.2.STR linkage analysis is consistent with SNP linkage analysis in the application of PGD.Because of its good polymorphism,abundant genetic information and low cost of detection,STR linkage analysis is more suitable for assisted reproductive technology.