Determination Of SMN1 And SMN2 Gene Copy Number In Spinal Muscular Atrophy By MALDI-TOF MS&Identification Of A Novel Deletion Mutation Causing α~0-thalassemia

Part Ⅰ Determination of SMN1 and SMN2 Gene Copy Number in Spinal Muscular Atrophy by MALDI-TOF MSBackground and objectives:Spinal muscular atrophy(SMA)is a fatal autosomal-recessive neuromuscular disease with a carrier frequency of～1 in 42 in Chinese population.Hence it’s of significance to conduct population screening and prenatal diagnosis of high-risk fetuses to prevent the birth of SMA babies and improve the population quality.The survival motor neuron gene 1(SMN1)is recognized as a causing gene while the deletion of modifier genes is associated with clinical phenotypes.As homozygous deletions of exon 7 and/or 8 of SMN1 are generally considered to be diagnostic for SMA and SMN2 proposes the important role in severity and type of disease as a modifier factor in different presentation of the disease,it’s of necessity to quantify the copy numbers of exon 7 of SMN1 and SMN2 to diagnose SMA patients,carriers and the normal clinically,and to predict the severity of the phenotype.Whereas,there are some flaws in current molecular diagnostic techniques to investigate copy number variations(CNVs)related to SMA,including time-wasting,high cost,low-throughput and low sensitivity,etc.Therefore,an accurate and sensitive assay based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)was established for the detection of SMN copy number in our study,in order to satisfy the demand of large-scale screening,and molecular diagnosis of SMA patients and carriers.Methods:Based on previous MALDI-TOF MS assay allowing the detection of point mutations of SMN1 and deletion mutations of modifying genes,this research was further optimized to quantify the copy numbers of SMN1 and SMN2.First of all,SMN genes and endogenous genes were simultaneously amplified by multiple PCR.Then the single base extension reaction was carried out after PCR products were treated with shrimp alkaline phosphatase to remove excess dNTPs.And SMN1,SMN2 and endogenous control locus were distinguished based on the differences in molecular weights by MALDI-TOF MS after the samples mixed with matrix.The copy numbers of SMN1 and SMN2 relative to endogenous genes were determined by comparing the peak area ratios between the test and control samples.And the test samples were genotyped according to the relative copy number of SMN1/2 and the copy number ratio of SMN1 and SMN2.When the novel assay was established and optimized,340 DNA samples with known genotypes were analyzed to evaluate its accuracy,7 samples with various genotypes to assess the sensitivity and 60 samples with one,two or three copies of SMN genes separately to estimate the repeatability.Finally,443 samples were detected in parallel with multiplex ligation-dependent probe amplification(MLPA)for clinical evaluation.Results:PCR system was preferably based on Agena reagents for the specificity and efficiency of amplified products,and the extension system containing double extension primers targeted to SMN gene was established for the consistent efficiency of SMN1 and SMN2.Additionally,a group of coefficients were determined to treat the measured copy numbers to narrow the gray regions among SMN genotypes.The novel MALDI-TOF MS assay for SMA was capable to accurately discriminate a range of copy numbers of SMN1 and SMN2.The system had high accuracy of 100%,good stability,high sensitivity(The detection limit reached 14 ng gDNA per reaction),and low cost.Furthermore,the results of 443 clinical samples in the wide screening were consistent with the MLPA results,suggesting the feasibility of the novel assay in the diagnosis of SMA.Conclusion:The new CNV analysis technology based on MALDI-TOF MS generally achieves the ration of SMA-related genes.And its accuracy,stability,sensitivity and high-throughput make it suitable for large-scale screening and clinical routine molecular testing.Moreover,our analytical strategy for the copy number may also serve as a reference for multi-gene CNVs detection.Part Ⅱ Identification of a Novel Deletion Mutation Causing α~0-thalassemiaBackground and objectives:Alpha-thalassemia is the world’s most common hemoglobin disorders,which is characterized by deficits in the synthesis of α-globin caused by α-globin gene’s mutations.Our study aimed to identify a case with a novelα-globin gene deletion,and analyse its influence on the expression of α-globin gene.Methods:The proband,a male newborn from Chenzhou City,Hunan Province of southern China,exhibited phenotype similar to α-thalassemia.Then the proband’s and his parents’ peripheral blood samples were collected to analyse hematological parameters.And their DNA samples were extracted to detect α-and β-globin genes by Gap-polymerase chain reaction(Gap-PCR),the reverse dot blot(RDB),Sanger sequencing and multiplex ligation-dependent probe amplification(MLPA).Further,targeted Next Generation Sequencing(NGS)was applied to more precisely locate the large deletion,and primers flanking both sides of the juxtaposition were designed to amplify the targets near both ends of the deletion by Gap-PCR and the breakpoints were verified by Sanger sequencing of the amplified products.Additionally,Real-time PCR was conducted to investigate the carrier(the father)’s α-globin mRNA level to analyse the effects of this mutation on gene expression.Results:Hematological phenotypic analysis indicated that the proband was a patient with α-thalassemia,his father was with thalassemia minor and his mother showed no obvious abnormality.A heterozygous deletion in the proband was identified by MLPA and NGS,which had proved to be of paternal origin.Gap-PCR and Sanger sequencing confirmed that the 71846 bp deletion with 5’ breakpoint within coordinates 188959 and 188970 and 3’ breakpoint between 260805 and 260816 was classified as α0-thalassemia.RNA analysis on the carrier revealed that the relative expression of HBA was lower than normal control,and almost parallel to other α0-thalassemia carriers.Conclusion:Our study identifies a case with a novel a-globin gene 71846 bp deletion that may be generated via non-allelic homologous recombination by targeted-NGS,and analyses its influence on the phenotype,which conduces to the enrichment of the mutation spectrum of α-thalassemia,and is of a certain reference significance in clinical detection and diagnosis of thalassemia.Also,it indicates that NGS is a useful tool in the location of unknown copy number variations.