Identification And Analysis Of Copy Number Variation And Splicing Mutation Of FBN1 Gene In Marfan Syndrome

Objective: Marfan syndrome(MFS)is a connective tissue disease involving multiple systems.Thoracic aortic aneurysm(TAA)is the most common life-threatening manifestation.We found two families with Marfan syndrome clinically.We screened,identified and analyzed the mutation of fibrillin 1(FBN1)gene of family members.We analyzed the pathogenesis of patients.In addition,we also made a clear diagnosis for patients in combination with genetic test,provided genetic counseling for family members and gave scientific suggestions on prevention and treatment.This study also makes a new supplement to the pathogenicity analysis of FBN1 mutation,and provides a new perspective for the etiology,diagnosis and treatment of MFS.Part 1: Identification and analysis of copy number variation of FBN1 gene in Marfan syndromeMethods: 1.Peripheral venous blood was extracted from 6 family members and genomic DNA was extracted.Whole exome sequencing(WES)was performed to screen for FBN1 gene mutation.2.Chromosome microarray analysis(CMA)was performed on blood samples to verify the results of WES further.Results: 1.the proband,his sister and daughter were screened a devo 0.76 Mb microdeletion on 15q21.1 through WES.The microdeletion co-segregated with the phenotypes in the family.2.CMA was performed on FBN1 of the proband and his family members.It verified WES results and confirmed the copy number variation(CNV).Part 2: Identification and analysis of splicing mutation of FBN1 gene in Marfan syndromeMethods:1.The aortic tissue was made into a pathological section and observed.2.Peripheral venous blood was extracted from two family members.Genomic DNA was extracted.WES was performed to screen for FBN1 gene mutation.3.RT-PCR was performed on the aortic tissue and observed through gel imaging.4.Sanger sequencing was performed to verify the WES results further.Results: 1.Pathological results showed that the fibrous tissue of the patient’s aortic valve proliferated,accompanied by myxoid change and glassy degeneration.2.The proband and his niece carried a splicing mutation c.4817-2a > G in FBN1 by WES,which was located on intron 39 of the g DNA(the second base from 5 ’to 3’).It is a heterozygous mutation.3.RT-PCR results showed that the proband had two PCR products.One is the same as the normal sample and the other is longer.The patient has an abnormal transcript.4.Sanger sequencing results showed that the normal PCR product was complete.The abnormal PCR product failed to obtain the peak map due to the low concentration.Conclusions: 1.The 0.76 Mb microdeletion on 15q21.1 is the pathogenic cause of the proband in the first family.This may be the minimum fragment CNV associated with MFS found so far.This study extends the gene mutation and phenotype spectrum of pathogenic CNV associated with MFS.2.FBN1 splicing mutation c.4817-2a > G is the pathogenic cause of the proband in the second family,which may cause the adjacent intron retention and eventually lead to MFS.The study needs to be further confirmed.At present,there are no reports about this splicing mutation.Our study enriches the gene mutation and phenotype spectrum of MFS.We speculate the possible pathogenic mechanism of this mutation,which lay a foundation for further research.