Quantitative Analysis Of SMN1 Gene And Carrier Testing Of Spianl Muscular Atrophy

Objective Using real time fluorescence quantitative PCR technology, to establish a stable, rapid and accurate approach which could be routinely used for determining survival motor neuronⅠ(SMN1) gene copy number, and use it to test carriers of spianl muscular atrophy (SMA), at the same time to assess the implemetion value of SMA carrier testing in genetic diagnosis and prenatal diagnosis of SMA.Methods We developed a quantitative test on the basis of TaqMan technology using specific minor groove binder (MGB) probes, meanwhile selecting exon 3 of factorⅧas an external reference locus that circumvents the problem of unavoidable deviations in DNA concentrations. Establish standard, then both standard and test samples were parallely amplified SMN1 gene andⅧfactor in different tubes, optimized the reaction condition and instituted a standard detecting protocol. Step-diluted standards were used to set up the standard curves of SMN1 gene andⅧfactor. According to the relation of the cvcle threshold and the standard curves, we could calculate the initial dose of SMN1 gene andⅧfactor of the sample DNA, and than got the copy numbers of SMN1 in sample DNA. To verify the reproducibility and stability of the RQ-PCR technology, four samples from male and female carriers and control persons, respectively were compared between and within batches; Meanwhile, we tested 48 cases of SMA patients confirmed by RFLP-PCR as well as 20 cases of putative carriers confirmed by linkage analysis to test the specificity and reliability of the assay. In this study we tested 200 cases of the general population,26 cases of SMA patients' parents and 16 cases fetus of prenatal diagnosis for SMA carriers testing. Results1. In the experiment of reproducibility and stability of the quantification approaches for the SMN1 gene, we get the range of measured SMN1 dosage between and within batches was [0.73-1.15] and [0.77-1.05] for the carriers, and for the control persons was [1.70-2.18] and [1.68-1.98], respectively; In the test of the specificity and reliability, of 48 cases SMA patients, all have 0 copy number of SMN1 gene, in the confirmed carrier group, the quantification range spans from 0.71 to 1.15, the mean copy number is 0.90±0.135.2. Screening of 200 control DNA samples revealed that four probands were carriers of only one SMN1 copy, the mean SMN1 dosage value was 0.88±0.162, which ranged from 0.75-1.05, thus corresponding to the range typical for certain carriers of the SMN1 deletion; The mean SMN1 copy number of 194 controls is 1.88±0.156, which ranged from 1.60-2.18, they are non-carriers; The other two probands carried are carriers of three SMN1 copies, the mean SMN1/factorⅧratio was 3.16±0.092. approximately 3.09 and 3.22, respectively.3. Of 26 SMA patients' parents cases, the measured SMN1 copy numbers is 0.91±0.163, ranged between 0.61 and 1.16 in 25 cases,96. 1% of individuals are carriers; Two copies were detected in 1 sample, the copy number is approximately 1.82.3.9% of individuals corresponding to double copies4. In the screening of 16 fetus samples, ten of them are non-carriers, the mean measured SMN1 copy number is 1.88±0.147, ranged from 1.61 to 2.11: Homozygous deletions of the SMN1 gene could be observed in the other 6 cases. the quantification range spans from 0.73 to 1.10. the mean copy number is 0.90±0.194.Conclusions1. The real time fluorescence quantitative PCR technique can be used for the quantification of the SMN1 gene.The result is accurate and reliable, simple and practical, and it can be automated and large-scale detection system.2. A clear-cut differentiation between the different copy number ranges could be observed in SMA carriers and non-carriers, so the SMN1 dosage analysis can be used for the carrier testing of SMA. 3. The implementation of SMA carrier testing has important scientific value and clinical significance in SMA genetic counseling, genetic risk assessment and genetic diagnosis, and also provided a major foundation for SMA gene diagnosis and prenatal diagnosis.