A Rapid NGS Strategy For Molecular Diagnosis And The Determination Of Unknown Pathogenic Genes Of Primary Spontaneous Pneumothorax

Part ? A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dube syndrome in patients with primary spontaneous pneumothoraxBackground:Primary spontaneous pneumothorax(PSP)or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome(BHDS)that is caused by heterozygous mutations in FLCN gene.Most of the mutations are SNVs and small indels,and there are also approximately 10%large intragenic deletions and duplications of the mutations.These molecular findings are generally obtained by disparate methods including Sanger sequencing and Multiple Ligation-dependent Probe Amplification in the clinical laboratory.In addition,as a genetically heterogeneous disorder,PSP may be caused by mutations in multiple genes include FBN1,COL3A1,CBS,SERPINA1 and TSC1/TSC2 genes.For differential diagnosis,these genes should also be screened which makes the diagnostic procedure more time-consuming and labor-intensive.Methods:40 PSP patients were divided into 2 groups.19 patients with different pathogenic mutations of FLCN previously identified by conventional Sanger sequencing and MLPA were included in test group,21 random PSP patients without any genetic screening were included in blinded sample group.7 PSP genes including FLCN,FBN1,COL3A1,CBS,SERPINA1 and TSC1/TSC2 were designed and enriched by Haloplex system,sequenced on a Miseq platform and analyzed in the 40 patients to evaluate the performance of the targeted-NGS method.Results:We demonstrated that the full spectrum of genes associated with pneumothorax including FLCN gene mutations can be identified simultaneously in multiplexed sequence data.Noteworthy,by our in-house copy number analysis of the sequence data,we could not only detect intragenic deletions,but also determine approximate deletion junctions simultaneously.Conclusions:NGS based Haloplex target enrichment technology is proved to be a rapid and cost-effective screening strategy for the comprehensive molecular diagnosis of BHDS in PSP patients,as it can replace Sanger sequencing and MLPA by simultaneously detecting exonic and intronic SNVs,small indels,large intragenic deletions and determining deletion junctions in PSP-related genes.Part ? Identification of a novel candidate gene for hereditary primary spontaneous pneumothoraxBackground:Primary spontaneous pneumothorax is a common emergency in department of thoracic surgery,most patients are adolescent or young onset,10%of the patients had a family history,although some were caused by mutations in the genetic syndrome,especially BHD can explain the cause of some patients,but at present,because disease and pathological mechanism of the disease are not clear.Our previous work makes us realize that unknown pathogenic genes responsible for familial spontaneous pneumothorax might exist.Methods:Clinical and genetic analysis were performed in PSP pedigrees.A pedigree of autosomal dominant inheritance was investigated and choosed,whole exome sequencing analysis were performed in the family members.Bioinformatics analysis,whole genome linkage analysis,predicted functional and expression analysis were also conducted to identify the candidate gene.To verify it,we also sequenced this gene in 100 PSP patients.Immunofluorescence of lung tissue was done to observe its expression and distribution.Results:Full spectrum of genetic analysis of a pneumothorax family,including linkage analysis and whole exome sequencing,were performed.By bioinformatics and pedigree analysis,we discovered a potential new pathogenic gene COL6A5 in the pedigree,and verified it by mutation detection in the seven other PSP patients.The expression of COL6A5 and the potential function of spectrum showed that it is a strong candidate gene of pneumothorax.Immunofluorescence of lung tissue showed that COL6A5 protein was expressed in alveolar septa,suggesting its importance in alveolar structure.Conclusions:A new pathogenic gene was proved to be associated with primary spontaneous pneumothorax.Further research on this gene need to be done to reveal the pathogenesis and molecular mechanism of bullae formation.