NGS, MLPA And Sanger Sequence–Based Study On Mutations Of FBN1

ObjectiveThe purpose of this study was to screen mutations in fibrillin-1（FBN1）,transforming growth factor beta receptor typeⅠ（TGFBR1） and transforming growth factor beta receptor typeⅡ（TGFBR2） genes on patients with Marfan syndrome（MFS） and Ectopia lentis syndrome（ELS） for investigating the pathogenesis of the MFS and ELS patients and and providing detecting bases for their family members’ presymptomatic diagnosis and their offsprings’ prenatal gene diagnosis.Methods1.The application of NGS and MLPA for gene diagnosis of MFS and ELS. Genomic DNA were extracted from whole blood sample of 22 MFS and 1 ELS patients. Each exon in FBN1 was screened with Next generation sequencing（NGS）, followed by PCR-Sanger sequencing with suggested mutation to confirm the results. Parents’ samples with no mutation identified were further analyzed by multiplex ligation-dependent probe amplification（MLPA） to screen large fragment mutation, followed by PCR-Sanger sequencing for suggested large fragment mutation by NGS and MLPA to verify the results.2. Analysis of a homozygous FBN1 mutations in a family with MFSGenomic DNA were extracted from peripheral blood of the family. Each exon in FBN1 was screened with NGS, followed by PCR-Sanger sequencing with suggested mutation to verify the results. Next Sanger sequence was performed on the proband’s family member. Subsequently we extracted the proband’s RNA from her aortic tissue, and analyzed the FBN1 transcript using RT-PCR followed by Sanger sequencing. Results1. The application of NGS and MLPA for gene diagnosis of MFS and ELS.A total of 20 FBN1 mutations in 20 out of 23 patients were identified, in which 11 were novel and 9 known. The mutations comprised 13 missense, 3 nonsense, 2 splice site, 1 frameshift, and 1 large fragment deletion. The 11 novel mutation were c.448T>C、c.1665C>A、deletion of exon18、c.3082+2T>C、c.3521T>C、c.4081T>G、c.5517C>A、c.6864₆871+1deletion CTGTGTAGG、c.6992G>A、c.7594 del A、and c.7713T>G. The remained 9 mutation were c.199T>C、c.640G>A、c.1585C>T、c.2413T>C、c.3037G>A、c.3380G>T、c.5497T>C、c.6424T>C and c.6451T>C. Further analysis by MLPA and PCR-Sanger sequencing indicated that the mutation of a large fragment deletion in a patient was c.2114-2357₂167+747del3158bp covering exon18 and its flanking areas. Nevertheless, mutations were not found either in TGFBR1 or in TGFBR2.2. Analysis of a homozygous FBN1 mutations in a family with MFS.The results demonstrated that the proband and her brother were homozygous for the splice site variant（c.7570+5G>A） in intron 61 of FBN1, while her parents and daughter were all heterozygous for the variant. She was born in a consanguineous family. By analyzing the FBN1 transcript using RT-PCR and Sanger sequencing, a deletion of exon 61 in FBN1 was detected.Conclusions1、FBN1 mutation may be the pathogenesis of the 19 MFS patients and the 1 ELS patients.2、The splicing mutation（c.7570+5G>A）in FBN1 may be the pathogenesis of the MFS homozygous family.