Identification And Analysis Of Two Rare Mutations Of FBN1 Gene In Marfan Syndrome

ObjectiveThe purpose of this study was to identify and analyse mutations of fibrillin-1(FBN1)in two patients with Marfan Syndrome(MFS)to clarify the pathogenic of the patient’s disorder,and to provide a basis for the pre-symptomatic detection of family members or the prenatal diagnosis,which they may need in future,and to offer a new supplement for the pathogenicity of FBN1 gene mutation and a new perspective for MFS gene diagnosis and gene therapy.Part 1: Identification and analysis of a synonymous mutation of FBN1 in a patient with Marfan SyndromeMethods1.Genomic DNA extracted from MFS patients and their family members were analyzed by using the Next generation sequencing technology(NGS)for screening FBN1 gene mutation before bioinformatics analysis.The found mutation was verified by Sanger sequencing.2.RNA was extracted from aortic tissue of the patients followed by RT-PCR and Sanger sequencing to show whether the mutation could cause an alternate splicing.3.The fragment in which the mutantion was located and its normal control were inserted to pcDNA3.1Myc-His C plasmid and transfected into Hela cells,from which the RNAs were extracted,and then the expression of the splicing products was analyzed by RT-PCR.Results 1.A heterozygous synonymous mutation(c.4773A>G)in exon 39 of FBN1 gene was screened out by NGS sequencing in a patient with MFS.The result of Sanger sequencing was consistent with it.Bioinformatics analysis presented that the synonymous mutation might cause the loss of the splicing site of 5’-tgtcctggagGGGAAGGTTT-3’ might further result in two kinds of exon skipping or intron retention.There was no change in predicted splicing site scores at both ends of exon 39.2.RT-PCR and Sanger sequencing for aortic tissue demonstrated skiping reading of exon 39 of FBN1 produced,which led expression of exon 38 directly linked to exon 40.3.Experiment of constructed recombinant plasmids transfected into Hela cells also showed exon 39 of FBN1 skipping which was consistent with the result of aortic tissue sequencing.Part 2: Identification and analysis of a large fragment deletion of FBN1 in a patient with Marfan Syndrome and her familyMethods1.DNA was extracted from the proband’s peripheral blood,and FBN1 gene mutation was screened with NGS.The mutation of large fragment deletion of FBN1 was confirmed by MLPA technique.2.The breakpoint of the mutation of large fragment deletion in FBN1 was further verified by long-fragment PCR followed by Sanger sequencing.ResultsThe results of NGS sequencing showed that exon 66 deletion in FBN1 gene and MLPA indicated the same deletion mutation.Subsequent Sanger sequencing displayed that a 1416 bp-deletion including former 511 bp of exon 66 and the last 905 bp of intron 65 of FBN1 gene.The proband’s sister and son had the same mutation.Conclusions1.Synonymous mutation of c.4773A>G in FBN1 gene was the pathogenic of the first case of MFS.The Synonymous mutation can lead to changes in its splicing site,which ultimately cause the exon 39 skip reading.2.Changes in splicing siteds caused by FBN1 gene synonymous mutations can lead to Marfan syndrome.3.Long fragment deletion of intron 65 and exon 66 of FBN1 gene is the cause of the second case MFS and her family.