Stduy On The Polymorphisms Of MTHFR Gene Exons In Simple Low Level Spina Bifida

Stduy on the Polymorphisms of MTHFR Gene Exons in Simple Low Level Spina BifidaPart one:The amplifications of MTHFR exons by PCR Objective:To observe the expression of MTHFR gene exons by Extracting the genomic DNAs and amplifying the exons(1,4,7,9)of MTHFR gene.Methods:Genomic DNAs were extracted from 144 blood samples by salting out method. Four exons(1,4,7,9) of MTHFR were amplified by PCR,templated by the genomic DNAs. PCR products were observed by agarose gel electrophoresis.Results:None of the samples was found have exonic deletion in the analysis.Conclusions:Exonic deletion was not found in the all of the samples including spina bifida patients and normal controls. Part two:The polymorphism analysises of MTHFR exons by SSCP methodObjective:To observe the polymorphism distributions of the 4 exons(1,4,7,9) in the test people and divide the people into different groups by polymorphisms for further identification.Methods:Four exons(1,4,7,9) of MTHFR were amplified by PCR,templated by the genomic DNAs. PCR products were observed by single strand conformation polymorphism(SSCP)method. According to the results,divide the samples into different groups for further identification.Results:Only one abnormal result was found in exon1,however,different kind of single nucleotide polymorphisms were found in the other 3 exons.Conclusions:The variations of SSCP electrophoresis suggested that there are diverse nucleotide constructions of the exons. Some of the mutations could be the dangerous factors in the development of spina bifida. Therefor, further investigation should be conducted to test whether the polymorphisms of MTHFR contribute to the cause of spina bifida. Part three:The restriction enzyme digestion and Direct DNA sequencing of MTHFR exonsObjective:To observe the different genotypes and the allele frequencies of the 4 exons(1,4,7,9)of MTHFR gene in the test people, and explore the possible roles of these polymorphisms in the development of spina bifida.Methods:The PCR products of exon 4 and exon 7 were digested by restriction enzyme and did polyacrylamide gel electrophoresi(sPAGE) to observe the polymorphisms of 677C→T and 1298A→C in these two exons. Direct DNA sequencing was carried out to identify the products of exon 1 and exon 9.Results:One synonymous mutation was found in the products of exon 1;The alleles frequencies in exon 4 and exon 7 had no significant difference between SB patients and normal controls,and there was also no significant difference between SB families and normal controls;A point intron mutation which located in the downstream of exon 9 was found in our study,and there was no significant difference in the test groups.Conclusions:The synonymous mutation of exon 1 did not affect the synthesization of MTHFR;The polymorphisms of 677C→T and 1298A→C in exon 4 and exon 7 have no relationship with the development of simple low level spina bifida in Han nationality population. The intron mutation downstream from exon 9 also has nothing to do with spina bifida.