Molecular Detection Of Spinal Muscular Atrophy By MALDI-TOF MS&Preimplantation Genetic Diagnosis Of α-thalassemia SEA Deletion Utilizing Whole Genome Amplification And Short Fragment Gap-PCR Method

Part Ⅰ Molecular detection of spinal muscular atrophy by MALDI-TOF-MSOBJECTIVE:Spinal muscular atrophy(SMA)is an autosomal recessive disorder with clinical phenotypic heterogeneity.It is reported that China currently has about 1,700～2,830 SMA patients born each year,and the number of SMA patients is about 30,000～50,000.Early diagnosis,prenatal diagnosis,and genetic counseling are of great significance in reduction of the number of SMA children born,improvement in the diagnosis and treatment of patients’ conditions,and decline the social economic burden.96.4%patients of SMA exhibit homozygous SMN1 deletion,and the remaining SMA are caused by compound variants,with one SMN1 deletion and one variant allele.SMN genes have similar structure and SMN2 only has 10%of the function of SMN1.But due to the effect of synthetic protein accumulation,SMN2 is an important modification gene of the SMA disease phenotype.According to reports,the NAIP,GTH2F2,and H4F5 genes are also associated with SMA disease.The methods for detecting SMA diseases include PCR-SSCP,PCR-RFLP,Real-time PCR,PCR-DHPLC,AS-PCR,MLPA and Sanger sequencing,NGS,etc.Because of the complexity gene structures,the heterogeneity of clinical phenotypes,and related locus function has not been clarified,these detection methods have certain technical defects,such as false negatives,high detection cost,cumbersome operation,time-consuming,and the need of professional bioinformatics,which are not suitable for large-scale sample screening,and fail to conduct a comprehensive test for mutation sites in Chinese population.Therefore,the disease lacks a molecular detection technology with high accuracy,easy operation and comprehensive location suitable for detection among Chinese population.Methods:Six high-frequency point mutations on SMN1(p.Ala2Val,p.Trp92Ser,p.Thr274Tyrfs*32,p.Tyr277Cys,p.Ser8Lysfs*23,p.Leu228*)and SMN1/2,NAIP,GTH2F2 and H4F5 genes were chosen as detection targets.Specific amplification primers and extension primers were designed for the target gene.After PCR,remove excess dNTPs,single base extension,analyzed by MALDI-TOF MS,the six mutation sites of the test sample were typed and the SMN1/2,NAIP,GTH2F2,and H4F5 genes’ copy number were determined.The method of the present study could be used to screen the SMA patients.Fistly,the detection for SMA by MALDI-TOF MS system was established and optimizated.Mutational plasmid DNA standard materials were constructed by the site-directed mutagenesis cloning to determine the detection ability in the system.The ability of system copy number detection was evaluated by blind analysis with 120 SMA samples that have been verified by MLPA.The sensitivity,repeatability and detection accuracy of the system were also evaluated.To evaluate the clinical application of the new method,120 ordinary human samples were screened and 24 samples were randomly selected for MLPA and Sanger sequencing verification.RESULTS:The established SMA MALDI-TOF MS system can accurately genotype 6 mutations on the SMN1,and also accurately determine whether SMN1/2,NAIP,GTF2H2,H4F5 gene copy number is 0 or not.The test results of 120 samples are consistent with the MLPA test results,and the accuracy rate can reach 100%.The system has good stability,high sensitivity,high accuracy,screening results for 120 small-scale populations by the new assay,which was consistent with the MLPA and the Sanger sequencing.Conclusion:A molecular detection method for SMA using MALDI-TOF-MS was established.It could be detected most common mutations on SMN1 and the quantitative detection of the copy number of pathogenic genes.Molecular diagnosis of most SMA patients has been achieved.The method is accurate,reliable,sensitive,low cost and comprehensive detection of genes,which is suitable for large-scale screening and clinical routine molecular testing.Part Ⅱ Preimplantation genetic diagnosis of a-thalassemia SEA deletion utilizing whole genome amplification and short fragment Gap-PCR methodObjective:Thalassemia is one of the most common autosomal recessive diseases,in the world.When the couple is of the SEA-α genotype,the fetus has a quarter chance of being severely a-thalassemia which can cause death in the womb or shortly after birth and severe obstetric complications in pregnant women.According to statistics,at least 2,600 pregnant women worldwide are at risk of gestating such fetuses each year,and approximately 6,600 affected fetuses are born.Preimplantation genetic diagnosis(PGD)can prevent and control the birth of children with severe a-thalassaemia from the source,avoiding the risk of pregnant women needing to take abortion or induce labor,greatly reducing pregnant women and their families’psychological burden.There are some reports on the pre-implantation genetic diagnosis method for thalassemia embryos,including MDA combined with short tandem repeat polymorphism analysis,Q-PCR with TaqMan probe,nested PCR combined with digital PCR for copy number variation analysis,etc.But some of them requires special equipment such as a sequencer or a digital PCR instrument,and others also need to perform family analysis,which is cumbersome and has a long detection time.The objective of the study is to develop a preimplantation genetic diagnosis system based on whole genome amplification(WGA)of blastocyst cells and short fragment Gap-PCR for α-thalassemia SEA deletion.Methods:Firstly,using the SEA carrier gDNA sample to establish and optimize the Q-PCR quality inspection system and the short segment Gap-PCR typing system.Evaluate the sensitivity,repeatability and accuracy of the detection system with different concentrations of gDNA.Then,20 samples from SEA carrier blood(2,3,4,5,6 cells,4 tubes each)were used to simulate blastocyst biopsies with different cell numbers.After the WGA of lymphoid samples by multiple displacement amplification(MDA),whose effect was examined by the Q-PCR system of the housekeeping gene GAPDH and β-actin.And the short-fagement Gap-PCR was used for a-Thalassemia SEA genotyping.Finally,12 clinical D5 blastocyst biopsy samples were collected and evaluated for clinical application.Results:Detection of lymphocyte samples with different cell numbers using the method developed in this study revealed no ADO in 3-cell samples,and the product quantity of WGA became stable for 4-cell samples.Of the 20 lymphocyte samples with different cell numbers,1 2-cell sample showed an allele drop-out(--SEA/--SEA),and the other 19 patients had the correct genotyping results(--SEA/aa).Of the 12 clinical blastocyst biopsies,2 failed WGA.The remaining 10 samples genotype were--SEA/αα(5 cases)and αα/αα(5 cases).The results were consistent with the actual genotypes.Conclusion:A preimplantation genetic diagnosis method for embryos with--SEA thalassemia by WGA combined with short-fragement Gap-PCR was established,which is rapid,accurate,and cost-effective and suitable for clinical routine applications.