Novel Methodology For Molecular Diagnosis Of Spinal Muscular Atrophy

Spinal muscular atrophy(SMA) is a common autosomal recessive neuromuscular disorder caused by degeneration of motor neurons in the anterior horn of the spinal cord. The degeneration process leads to progressive symmetric muscle weakness(more proximal than distal) associated with muscle atrophy.The frequency of the disease is calculated to be approximately 1:10000 with a carrier frequency of 1:50.Three clinical types of the disease have been defined according to the age of onset and decreasing severity of the disease.The SMA disease-determining gene SMN1 and its homologue SMN2 were found to lie within the telometric and centrometric halves,respectively,of a large inverted repeat on chromosome 5q13.The difference between SMN1 and SMN2 lies in exon 7 in the coding region.Since about 95%of SMA patients have homozygous deletion of exon 7 of SMN1, testing by PCR-RFLP for detecting homozygous SMN1 loss is preferred for molecular diagnosis and prenatal molecular diagnosis of SMA.In the first part in our study,two methods,whole blood PCR-RFLP and allele-specific PCR,both of which were quicker than the conventional PCR-RFLP method,were established and applied in the molecular diagnosis of 14 SMA patients.Th results of the two methods showed all the patients had homozygous deletion of SMN1 exon 7,coinciding with that of the conventional PCR-RFLP method.The two methods,whole blood PCR-RFLP and allele-specific PCR,might be of value in the rapid molecular diagnosis of SMA.The second part presents a novel strategy for prenatal molecular diagnosis of SMA, and it was applied in the prenatal molecular diagnosis for 13 fetuses of 11 Chinese SMA families.DNA was extracted from amniotic fluid cells before and/or after culture.STR profiling was carried out to evaluate the contamination of amniotic DNA by maternal genomic DNA.Two methods,PCR-RFLP and allele-specific PCR,were used to analyze exon 7 of SMN gene from amniotic DNA.The results showed that there was no contamination of amniotic DNA by maternal genomic DNA.In conventional PCR-RFLP. part of the PCR product(189bp) from amniotic DNA of 9 fetuses(fetuses A,C,D,E-2, F-1,F-2,I,J and K) remained intact after digestion with DraI,while the PCR product from amniotic DNA of the remaining 4 fetuses(fetuses B,E-1,G and H) was completely digested by DraI.In allele-specific PCR,exon 7 of both SMN1 and SMN2 could be seen when amniotic DNA of the 9 fetuses(fetuses A,C,D,E-2,F-1,F-2,I,J and K) was used, while only exon 7 of SMN2 could be seen when amniotic DNA of the remaining 4 fetuses(fetuses B,E-1,G and H) was used.In conclusion,Homozygous deletion of SMN1 was not detected in fetuses A,C,D,E-2,F-1,F-2,I,J and K,while homozygous deletion of SMN1 was present in fetuses B,E-1,G and H,predicting extremely high risk of developing SMA after birth.The novel strategy was reliable and efficient for prenatal molecular diagnosis of childhood-onset SMA.Since about 95%of SMA patients have a homozygous deletion of exon 7 of SMN1,a PCR-RFLP testing,which was refered to as the standard diagnostic test,is suitable for detecting homozygous SMN1 loss.However,this method cannot detect heterozygous SMN1 loss,and cannot distingush SMA carriers from non-carriers.Therefore,quantitative methods should be developed to solve those problems.In the third part in our study,we introduced MLPA technique in the molecular diagnosis of SMA,which allowed not only the detection of homozygous deletion of SMN1 gene but also the determination of gene dosage of SMN1 and SMN2.The genomic DNA was isolated from peripheral blood of 13 SMA patients(9 belonged toâ… type,1 belonged toâ…¡type,3 belonged toâ…¢type) in which the loss of both SMN1 copies had been previously confirmed by conventional PCR-RFLP and allele-specific PCR,31 parents of SMA patients,50 healthy individuals unaffected with SMA and with no family history of SMA, and prenatal samples(amniocytes,before and /or after culture) of 10 fetuses from those SMA families.All of the genomic DNA was analyzed by MLPA,and the ratio(relative copy number) and absolute copy number of SMN1 and SMN2 gene of each sample were obtained by data analysis using manual Excel sheets.At the same time,the results of homozygous deletion analysis of SMN1 gene by MLPA were compared to those by conventional PCR-RFLP and allele-specific PCR.The analysis of MLPA showed that,for SMN1 gene,all of the 13 patients had homozygous deletion of SMN1,which was in complete agreement with the results of conventional PCR-RFLP and allele-specific PCR. Of 31 parents,2 each had 2 copies,29 each had 1 copy.Of 50 healthy individuals,1 had 1 copy,1 had 3 copies,48 each had 2 copies.Of 10 fetuses,2 had homozygous deletion of SMN1,which was in complete agreement with the results of conventional PCR-RFLP and allele-specific PCR,5 each had 1 copyn,and 3 each had 2 copies.For SMN2 gene,2 typeâ… patients each had 2 copies.7 typeâ… patients each had 3 copies,1 typeâ…¡patient had 3 copies, 3 typeâ…¢patients each had 4 copies.Of 31 parents,5 each had 1 copy,12 each had 2 copies,14 each had 3 copies.Of 50 healthy individuals,2 each had homozygous deletion of SMN2,6 each had 1 copy,42 each had 2 copies.Of 10 fetuses,9 each had 2 copies,1 had 3 copies.The MLPA technique had proved to be a robust and reliable tool for the molecular diagnosis of SMA.Approximately 5%of SMA patients are known to have no homozygous SMN1 deletion,which are believed to have a subtle mutation in one or extremely rarely in both SMN1 alleles instead.In the fourth part in our study,we present a complete mutation detection system of SMN1 gene,performing mutation analysis and describing the genotype of a patient and his family.Deletion analysis of SMN1 exon 7 by the conventional PCR-RFLP and allele-specific PCR,and SMN1 and SMN2 gene dosage by MLPA were performed for the patient and his parents.RT-PCR and sequencing was performed,the RT-PCR amplification product of the patient was cloned into TA cloning vector and multiple subclones were sequenced directly;the PCR of SMN exon 5 from the genomic DNA of the parents and direct sequencing were performed to comfirm the mutation.In deletion analysis of SMN1 exon 7,no homozygous deletion of SMN1 was observed in the family;the gene dosage analysis by MLPA showed that the patient had 1 copy of SMN1 and 1 copy of SMN2,the father had 2 copies of SMN1 and 2 copies of SMN2,and the mother had 1 copy of SMN1, lacking SMN2.A previously unreported missense mutation,i.e.S230L,was identified from the patient by RT-PCR,subcloning and sequencing,and this mutation allele was also found in the father by PCR and direct sequencing.Results of genotyping showed that the patient had 1 copy of SMN1 in which the S230L mutation was identified and 1 copy of SMN2,the father had 2 copies of SMN1(one of which carried the S230L mutation) and 2 copies of SMN2,and the mother had 1 copy of SMN1,lacking SMN2.In conclution,the comprehensive testing methods for molecular diagnosis and prenatal molecular diagnosis of SMA,including deletion analysis of SMN1,dosage analysis of SMN1 and SMN2 gene,and subtle mutation analysis of SMN1 gene,offered complete evaluation of SMA patients and their families.