A Preliminary Study On The Copy Number Characteristics Of Survival Motor Neuron Gene (SMN)

BackgroundSpinal muscular atrophy（SMA）is one of the most common genetic causes of death in children two years old,with a population incidence of approximately 1/10000 to 1/6000,and the average carrier ratio of normal population is 1/40.The clinical manifestation of SMA is to carry out sexual muscle weakness,muscle atrophy,etc.Severe cases can die of breathing failure caused by respiratory muscles.The pathogenic gene of SMA is survival motor neuron（SMN）gene,which contains two highly homologous genes,namely SMN1（survival motor neuron 1）and SMN2（survival motor neuron 2）gene.Homozygous deletion or compound heterozygous mutation of SMN1 gene can cause SMA,while SMN2 gene as a modifier gene,it is related to the severity of SMA disease^[1].At present,SMA is incurable,and the prevention of birth defects in children with SMA is mainly based on prenatal diagnosis.Commonly used clinical detection techniques include multiplex ligation-dependent probe amplification technology（MLPA）,real-time fluorescent PCR technology,etc.Among them,MLPA technology is widely used in clinical applications,but it is costly and time-consuming.Therefore,carrying out a convenient,cheap and accurate new technology of SMA carrier screening and prenatal diagnosis is the most effective preventive means to reduce the birth of SMA children,and it is also the difficulty and hot spot of molecular genetic testing of prenatal diagnosis.In this study,We tried to use a convenient and inexpensive fluorescent survival motor neuron gene copy number detection kit（fluorescence PCR-capillary electrophoresis）to detect the SMN copy numbers,to clarify the accuracy of the fluorescence PCR-capillary electrophoresis for SMA,and use this technology to preliminary analysis and evaluate the SMN gene copy numbers of 2011 pregnant women in Henan region.Objective1.The validity and accuracy of a new survival motor neuron gene copy number detection kit were evaluated by using MLPA as control method,in order to provide a fast,efficient and accurate molecular diagnostic technique for SMN1 and SMN2 gene copy number detection.2.The copy numbers of SMN1 and SMN2 gene in 2011 pregnant women in Henan region was studied to obtain the frequencies of different alleles of SMN1 and SMN2 in this population,so as to preliminarily estimate the distribution characteristics of SMA carriers in Henan population,and to provide preliminary basic data for prenatal screening and prenatal diagnosis strategy formulation of SMA in Henan population.Methods1.The 656 DNA samples from our center were selected as the study samples.MLPA and the survival motor neuron gene copy number detection kit（fluorescence PCR-capillary electrophoresis）were used for simultaneous detection,and the consistency of the test results of the two methods was compared.2.The 2011 pregnant women in our hospital were randomly selected according to regional distribution in Henan province.The copy numbers of SMN1 and SMN2genes in their peripheral blood was tested by fluorescence PCR-capillary electrophoresis,and the distribution characteristics of copy number of SMN1 and SMN2 genes and the frequency characteristics of different alleles of SMN1 and SMN2were analyzed.3.The statistical software IBM SPSS Statistics 23.0 was used to carry on the chi-square test for the counting data,P<0.05.The difference was statistically significant.Result1.In 656 DNA samples,the results of MLPA and fluorescence PCR-capillary electrophoresis were identical（100%）.Among them,19 cases（2.89%）were detected when the total copy numbers of SMN1 and SMN2 were 5 or more,and the total copy numbers were not more than 6.No more than 4 copies of SMN1 and SMN2 were detected.28 cases（4.26%）were detected in samples with 3 copies or more of SMN1gene.16 cases（2.44%）were detected in samples with 3 or more copies of SMN2gene.2.Among the 2011 peripheral blood samples,genomic DNA extraction failed in10 samples,and 18 cases（0.90%）had one copy of SMN1.A total of 14 SMN genotypes were detected.Among them,two genotypes（607 cases of SMN1:SMN2=2:1,1158 cases of SMN1:SMN2=2:2）accounted for the highest proportion,and the detection rate of population was 88.20%.3.Among the 2001 valid samples,85 cases（4.25%）were detected when the total copy numbers of SMN1 and SMN2 were 5 copies or more,and the total copy numbers were not more than 6.Among them,78 cases had 5 copies of SMN1 and SMN2（3.90%）.4.Among the 2001 valid samples,the total copy numbers of SMN1 gene were not more than 4,and 90 cases（4.50%）had 3 copies or 4 copies.The total copy numbers of SMN2 gene was not more than 4,and 64 cases（3.20%）with 3 copies or 4copies.5.The alleles of SMN2 were named according to the copy number of SMN2 gene on each chromosome according to the naming method of SMN1 alleles,which were named as SMN2-0（chromosome with 0 copy number of SMN2 gene）,SMN2-1（chromosome with 1 copy number of SMN2 gene）,and SMN2-2（chromosome with 2copy numbers of SMN2 gene）.Based on the copy number distribution of SMN2 in2001 samples,the allele frequencies of SMN2-0,SMN2-1 and SMN2-2 were estimated to be 20.71%,77.66%and 1.62%,respectively.According to the above allele frequency,Hardy-Weinberg genetic balance test was performed in the study population,P>0.05.6.The frequency of alleles 1 and 2 of SMN1 gene was estimated to be 97.23%and 2.32%,respectively,based on the distribution characteristics of copy number of SMN1 gene in 2001 valid samples.Conclusions1.The fluorescence PCR-capillary electrophoresis is an effective tool for the detection of copy numbers of SMN1 and SMN2 genes,and the detection effect of the target gene copy number is equivalent to MLPA,but the operation is simpler and cheaper.2.The copy number characteristics of SMN1 and SMN2 in 2011 pregnant women in Henan were preliminarily defined.The total copy numbers of SMN1 and SMN2were less than or equal to 6.The gene copy numbers of SMN1 and SMN2 were less than or equal to 4.SMN1 and SMN2 gene had significant copy number variation.3.In 2011 samples,the proportion of those with 1 copy of SMN1 was significantly lower than the proportion of SMA carriers reported in the literature,and the gene frequency of allele 2 of SMN1 in the study samples was as high as 2.32%,so it was speculated that there might be a certain proportion of[2+0]in the study samples.