Marfan Syndrome And Bronchodilation Pathogenic Mutation Studies

Part 1 Application of NGS in screening for pathogenic mutations in 125 Chinese patients with Marfan syndromeMarfan syndrome(MFS)is a pleiotropic connective tissue disease inherited as an autosomal dominant trait,due to mutations in the FBN1 gene encoding fibrillin 1.It is an important protein of the extracellular matrix that contributes to the final structure of a microfibril.Few cases displaying an autosomal recessive transmission are reported in the world.The FBN1 gene,which is made of 66 exons,is located on chromosome 15q21.1.Objective:Study the mutation spectrum of Chinese MFS;The MFS genotype-phenotypic correlation analysis;Identify new pathogenic genes responsible for MFS.Subjects and methods:1.Subjects:125 Chinese patients with MFS and their family members;2.Methods:Ion torrent PGM sequencing:125 patients were sequenced by Ion torrent PGM sequencing platform.Linkage analysis:STR markers near the FBN1 gene of the MFS families were genotyped and linkage analysis was performed.For the families showling linkage with FBN1,the FBN1 gene exons were re-sequenced.For those not linked with FBN1,whole exome sequencing was performed.A twin MFS family was performed exome sequencing.MLPA:MLPA(Multiples ligation-dependent probe amplification)was performed in patients for whom the pathogenic mutations were not detected by PGM sequencing;qPCR was performd to shorten the upstream and downstream flanking sequence of the breakpoint;Long-Range PCR was performed for breakpoints flanking sequence amplification;Total RNA was extracted from aortic tissue of the patient with FBN1 exon 48-53 deletion,cDNA synthesis and transcription level analysis were performed.Genotype-phenotypic correlation analysis was performed.Results:Ion torrent PGM sequencing results:In 96 out of 125 patients,100(likely)pathogenic variants were identified with 55 novel.97(97.0%)pathogenic mutations were located in the FBN1 gene,2(2.0%)were located in the TGFBR1 gene,and one(1%)in TGFBR2.Amone 100 variants,55(55%)are missense mutations,11(11.0%)are splice site mutations,11(11.0%)are frameshift mutations and 15(15.0%)are nonsense mutations.Unexpectedly,of 125 patients,one patient(0.8%)was compound heterozygous and 2 patients(1.6%)had 2 variants in one fibrillin-1(FBN1)allele.In family 50,the linkage analysis showed tight linkage with the FBN1 gene,and a frameshift deletion mutation was found bysequencing the entire FBN1 gene.Family 58 didn't show linkage withFBN1,and a SMAD3 mutation was detected.Four gross deletions were identified in FBN1:exon 6,exons 48-53,exons 49-50,and exons 1-36 deletions.Three deletions(exon 6,exons 48-53,and exons 49-50)were predicted to be in-frame deletions;the remaining deletion(exons 1-36)was a out-of-frame deletion and nonsense mediated mRNA decay was expectedso that loss of one allele of the FBN1 gene will be predicted.The breakpoints of these 4 deletions were cloned,and sequencing of the fragments junctions revealed deleted sizes of 16,551 base pairs(bp),10,346 bp,4,563 bp,and 187,067 bp respectively.By analyzing the transcription levels from patients'RNA samples available,the deletion of exons 48-53 was confirmed to be in-frame.This deletion removed several calcium-binding EGF domain(s)and/or TB domain(s)of fibrillin-1.Clinical features of three patients with in-frame deletions differed from those of one patient with exonl deletion of FBN1.Genotype-phenotypic correlation analysis:The clinical features were characterized and compared,and we found that the patients with splice site mutations and those with mutations involving cysteines had a higher prevalence of ectopia lentis;aortic dissection occurred more frequently in patients with protein-truncating mutations.The compound heterozygous patients and patients with double variants in one FBN1 allele exhibited mild classical MFS features.Patient with p.C123G are associated with rare common carotid artery dissection.Genotype-phenotype analysis in patients with large deletions,gross deletions of FBN1 exons results in variable phenotypes of MFS,but tends to cause severe Neonatal MFS.Conclusion:In this study,55 novel mutations were detected and that enriched the mutation spectrum of the MFS.By Genotype-phenotype correlation analysis,we found that the patients with splice site mutations and cysteine mutations were more likely to suffer lens ectopic than those patients with PTC(truncating mutation).Patients with PTC mutations were more likely to have aortic dissection phenotype.MLPA is an effective method for detecting large deletions in MFS patients.Patients with in-frame deletion mutations tend to suffer severe Neonatal MFS.Haploinsufficiency of FBN1 is responsible for the phenotypes in classic MFS.Compound heterozygous mutation can lead to MFS,which has important implications for prenatal screening of Marfan syndrome.Part 2 Next-generation sequencing for identifying genetic variants in Chinese adults with bronchiectasisDefective host-defense,including impaired ciliary function or ultrastructure and mucus clearance,has been associated with bronchiectasis.However,whether genetic mutations related to host-defense defects(e.g.altered mucus properties,ciliary defects)are implicated in bronchiectasis pathogenesis remains unclear.Objective:To reveal the genetic susceptibility in patients with bronchiectasis,and to provide a new standard for the etiological classification of bronchiectasis.Subjects and methods:Subjects:Experimental study of 192 patients with bronchiectasis in China and 100 individuals without lung disease as normal control.Methods:Ion torrent PGM sequencing:192 patients and 100 normal controls were sequenced by Ion torrent PGM sequencing platform.(TG)m Tn repeats genotyping:intron 8 of CFTR was amplified by PCR and analyzed by ABI3730 DNA Analyzer in 192 patients and 100 normal controls.Analysis of mutations in different genes:analysis compound heterozygous mutations of CFTR,trans-heterozygotes for CFTR/ENaC mutations,CFTR gene V470M polymorphism;PCD-related pathogenic homozygous and compound heterozygous mutations;Genotype-Phenotype correlation analysis:the patients with DNAH5,DNAH11,CFTR and CFTR/ENaC recessive pathogenic mutations were analyzed.Investigation of the etiology of patients with pathogenic mutations and study of the role of genetic factors in the pathogenesis of bronchiectasis.Results:1.In this study,192 cases of adult patients with bronchiectasis were classified by the etiology,the most common one is post-infectious(27.6%).2.IVS8 5T were found in 13 patients,one patient with homozygous IVS8 5T,and 9 individuals carry IVS8 5T in.normal control.3.In the CFTR and ENaC gene,a total of 3 patients were found to have two mutations in CFTR,2 patients carry CFTR/ENaC trans-heterozygotes mutations;38 patients are homozygous for M470,12 of them were found to carry another CFTR gene mutations,which is significantly higher than that in normal controls(6.3%vs.1.0%,P=0.039);Twenty patients were found to carry two mutations in PCD related pathogenic genes(10.4%,20/192).14 cases carry DNAH11 mutations,4 cases carry DNAH5 mutations,one carries CCDC40 gene mutation,one carries HEATR2 homozygous mutation and 1 carries RPGR(X chromosome)mutation.Of the 100 normal subjects,3 were found to carry two DNAH11 mutations.4.Patients with two DNAH5 mutations were found to have a severe phenotype compared to patients with DNAH11 mutations(BSI mean:8.8 vs.4.2);patients with trans-heterozygotes for CFTR/ENaC mutations have more severe phenotype than those patients carrying two CFTR gene mutations(CFTR/ENaC BSI=10 vs CFTR/CFTR BSI = 5.2).5.A total of 26 patients were found to carry pathogenic mutations,which accounted for 13.54%of the total number of patients.They can be classified as following according to different etiology:16 idiopathic,5 post-tuberculosis,2 gastroesophageal reflux,11gG1 deficiency,1 asthma and 1 measles.Conclusions:We have identified mutations mainly associated with CF and PCD in Chinese bronchiectasis patients.Homozygous M470 variant plus other CFTR mutants predispose to bronchiectasis.Some mutations(particularly DNAH5 mutation)are linked to greater bronchiectasis severity.A total of 13.54%of the patients were affected by genetic factors.This study provides a new standard for classification of bronchiectasis by etiology.