Exome Sequencing And Multi-Omics Studies Of Bronchiectasis

Background: Bronchiectasis is a chronic infectious disease characterized by destruction of lung structure,persistent dilatation of the airways and repeated respiratory infection.The clinical heterogeneity observed in bronchiectasis is considerable.Despite conducting a thorough examination,the majority of patients are classified as having idiopathic bronchiectasis due to the unknown etiology.The determination of the etiology of hereditary bronchiectasis can be achieved through whole exome sequencing.The pathogenesis of bronchiectasis is influenced significantly by chronic infection and host inflammation.However,in some patients with substantial amounts of purulent sputum,the etiology of chronic infection remains elusive,as sputum bacterial culture yields repeated negative results.The interaction between host and microorganisms and the serum metabolic characteristics of bronchiectasis may also be involved in the pathophysiological process of bronchiectasis and related to the severity of the disease,but there is a lack of related research at present.Objective: This study aims to investigate: 1)the clinical characteristics of hereditary bronchiectasis,its genotypes and the molecular characteristics;2)the respiratory pathogen spectrum in bronchiectasis and the association between different pathogen infections and clinical features;3)The interplay between microbiome and the host transcriptome in bronchiectasis.4)differences in serum metabonomics in patients with bronchiectasis,and potential biomarkers that correlated with clinical features.Methods:1.Retrospective analysis of whole exome sequencing data was performed on patients with idiopathic bronchiectasis at Second Xiangya Hospital of Central South University from January 2015 to December2022.A primary ciliary dyskinesia(PCD)diagnostic platform was established,which included various tests such as the measurement of nasal nitric oxide concentration,semen analysis and Papanicolaou staining,high-speed video microscopy analysis of the cilia,immunofluorescence of respiratory cilia and sperm,transmission electron microscopy of cilia,and whole exome sequencing.The clinical characteristics,respiratory cilia structure,and function of the patients were evaluated,and the association between different PCD genotypes and phenotypes was studied.The prognosis of patients with hereditary bronchiectasis was also followed up.2.A prospective study was conducted,enrolling stable bronchiectasis patients who visited the outpatient clinic of Xiangya Second Hospital of Central South University from October 2020 to August 2022.The characteristics and quality of sputum were assessed using the Murray sputum purulence score color card and Gram stain examination of sputum smears.Sputum samples meeting the criteria for purulent sputum were subjected to bacterial culture,as well as DNA and RNA metagenomic next-generation sequencing(mNGS).The results of DNA and RNA mNGS detection were compared with bacterial culture,and the negative bacterial culture results were analyzed.Furthermore,the correlation between mixed bacterial infections and clinical indicators was investigated.3.A retrospective analysis was conducted on the sputum metagenomic next-generation sequencing(mNGS)data of stable bronchiectasis patients who visited the outpatient department of Second Xiangya Hospital of Central South University from October 2020 to August 2022.The relative abundance of the microbiome at the phylum and genus levels in bronchiectasis patients was analyzed.The overall composition of the microbiome in patients with different severities of bronchiectasis was compared using principal coordinate analysis and permutational multivariate analysis of variance.The random forest method was employed to identify microbiota associated with disease severity.Additionally,using the RNA sequencing data of host transcripts from mNGS,differential gene expression analysis was performed to identify genes differentially expressed in sputum host transcriptomes of patients with varying disease severity,thus identifying enriched signaling pathways of these differentially expressed genes.A microbiome-host transcriptome interaction analysis based on generalized boosted linear models was employed to identify the association network between severity-related microbiota and differentially expressed genes.4.A prospective study was conducted from October 2022 to January2023,enrolling bronchiectasis patients and healthy controls who visited Second Xiangya Hospital of Central South University.Serum samples were collected from both bronchiectasis and healthy controls.Liquid chromatography-mass spectrometry(LC-MS)technology was utilized to perform untargeted metabolomics analysis on the serum samples of bronchiectasis patients and healthy controls.By employing orthogonal partial least squares discriminant analysis(OPLS-DA),differential metabolites between bronchiectasis and healthy controls were identified.Additionally,weighted gene co-expression network analysis(WGCNA)was integrated to identify key differential modules and metabolites related to clinical indicators.Results:1.The clinical and molecular characteristics of the hereditary bronchiectasis cohort(1)A total of 322 cases of idiopathic bronchiectasis with available exome sequencing results were included in the analysis.Based on the combined results of ciliary structure and functional testing,a total of 77 cases of hereditary bronchiectasis were diagnosed.Among them,55 cases were classified as primary ciliary dyskinesia(PCD),18 cases as primary immunodeficiency diseases,3 cases as cystic fibrosis,and 1 case as Young’s syndrome.Among the remaining 245 patients without a clear etiology,15 cases were found to have a high likelihood of PCD through the PCD diagnostic platform.However,currently,no clinical testing methods can definitively confirm the diagnosis in these cases.(2)Among the 55 confirmed PCD patients,two cases showed negative results in exome sequencing but were diagnosed based on transmission electron microscopy of cilia.By combining measurements of nasal nitric oxide,RNA splicing validation,high-speed video microscopy imaging of cilia,and ciliary immunofluorescence analysis,56 pathogenic or potentially pathogenic variants in 17 genes were identified in 53 patients.The most common gene types observed were DNAH11(14/53,26.4%),DNAH5(5/53,9.4%),RSPH4A(5/53,9.4%),CCDC40(4/53,7.5%),DNAAF4(4/53,7.5%),CCNO(3/53,5.7%),and ODAD3(3/53,5.7%).(3)Comparing the clinical features among common PCD genotypes,significant differences were observed in visceral situs inversus(P=0.003),olfactory dysfunction(P=0.006),lung atelectasis(P=0.010),and infertility(P<0.001)between patients with different genotypes.Male patients with DNAH5 variants(n=3)and female patients with DNAH11variants(n=10)exhibited normal reproductive capacity.On the other hand,female patients with RSPH4 A variants(n=3),CCDC40 variants(n=2),DNAAF4 variants(n=2),and DNAAF2 variants(n=2)were all infertile.(4)After a median follow-up of 4 years,6 patients with PCD,1patient with primary immunodeficiency,1 patient with cystic fibrosis,and1 patient with a high likelihood of PCD(based on low nasal nitric oxide concentration,abnormal ciliary rotational oscillation,and negative exome sequencing)died due to pulmonary infection and respiratory failure.The median age at death was 31 years(interquartile range,28-35;range,19-55).2.The pathogens of sputum in stable bronchiectasis patients(1)A total of 59 patients with stable bronchiectasis,as determined by a Murray sputum purulence score of mucopurulent or purulent,were included in the study.The mNGS results of all purulent sputum samples revealed the presence of bacterial infections.Additionally,low-level Candida albicans was detected in 9 cases,respiratory RNA viruses in 6 cases,and other herpesviruses.The most common bacteria identified were Pseudomonas aeruginosa(35/59,59.3%),Haemophilus influenzae(13/59,22.0%),Moraxella catarrhalis(7/59,11.9%),Streptococcus pneumoniae(5/59,8.5%),Staphylococcus aureus(5/59,8.5%),Escherichia coli(3/59,5.1%),and Klebsiella pneumoniae(3/59,5.1%).(2)There was a high concordance between the bacterial detection results of DNA mNGS and RNA mNGS,with only two cases showing false-negative results for Staphylococcus aureus and Moraxella catarrhalis in RNA mNGS.The number of bacterial-specific aligned sequences in DNA mNGS was significantly higher than in RNA mNGS(P<0.001).However,RNA mNGS detected respiratory RNA viruses that were not detected by DNA mNGS,including human rhinovirus,enterovirus,and human coronavirus.(3)The positive rate of sputum culture was 83.1%(49/59).m NGS detected bacterial infections in all patients,and in the 10 cases where bacterial culture did not fully detect the presence of bacteria,mixed bacterial infections were discovered.The bacteria that were negative in sputum culture include: bacteria that do not grow using conventional cultivation methods,such as Nocardia cyriacigeorgica and Mycobacterium tuberculosis;common fastidious bacteria that may yield false negatives in sputum culture,such as Streptococcus pneumoniae,Haemophilus influenzae,and Moraxella catarrhalis;anaerobic bacteria such as Streptococcus constellatus and other anaerobes;and mucoid Pseudomonas aeruginosa,which exhibits slower in vitro growth and requires extended culture and observation time.(4)Among stable patients with bronchiectasis,23.7%(14/59)experienced mixed bacterial infections involving at least two types of bacteria.Patients with multiple bacterial infections exhibited more severe decline in pulmonary function parameters compared to those with single bacterial infections.Their forced expiratory volume in one second(FEV1)was lower,with median values of 1.4L and 1.8L respectively(P=0.040).The FEV1% predicted values were also smaller,with median values of52.9% and 68.8% respectively(P=0.010).Additionally,there was a significant difference in the FEV1/forced vital capacity(FVC)ratio,with median values of 63.6% and 72.0% respectively(P=0.010).3.The interplay between the sputum microbiome and the host transcriptome in stable bronchiectasis patients(1)In the sputum microbiome of stable bronchiectasis patients,the predominant bacterial phyla at the kingdom level include Proteobacteria,Firmicutes,and Actinobacteria.At the genus level,the dominant genera are Pseudomonas,Streptococcus,Rothia,Haemophilus,and Neisseria.(2)Principal Coordinate Analysis(PCoA)revealed significant differences in microbial composition between patients with mild-to-moderate bronchiectasis(Bronchiectasis Severity Index,BSI < 9)and those with severe bronchiectasis(BSI ≥ 9)(Per MANOVA test,P=0.014).Random Forest analysis,based on the average Gini coefficient reduction,identified several microbial genera associated with severe bronchiectasis,including Pseudomonas,Haemophilus,Moraxella,Klebsiella,and Treponema.On the other hand,genera such as Mycobacterium,Neisseria,and Abiotrophia were found to be associated with milder bronchiectasis.(3)Transcriptomic analysis revealed that the majority of genes in the sputum of severe bronchiectasis patients are upregulated.Differential gene expression analysis between patients with different severity levels primarily identified enrichment of genes involved in immune response-related signaling pathways,such as neutrophil extracellular trap formation,chemokine signaling pathways,leukocyte transendothelial migration pathways.(4)Network analysis of the microbiome-host interactions revealed that the genus Neisseria may be associated with decreased expression of PLEK and PROK2 genes in the host,indicating a potential link to reduced inflammation and mild-to-moderate bronchiectasis.On the other hand,the genus Moraxella may be associated with increased expression of the CCL3 gene in the host,suggesting a potential association with heightened inflammation and severe bronchiectasis.4.Serum untargeted metabolomics in patients with bronchiectasis(1)A total of 70 patients with bronchiectasis and 10 healthy controls were included in the serum untargeted metabolomics study.After excluding one outlier sample,a total of 1919 metabolites were detected.(2)When comparing bronchiectasis patients to healthy controls,it was observed that 82 metabolites had variable importance values ≥1and showed significant intergroup differences with a P-value <0.05.Several of these differing metabolites were found to be associated with oxidative stress.(3)WGCNA identified that the black and turquoise modules were significantly associated with lung function,radiological severity assessed by Reiff score,or BSI score(P<0.05).(4)The differentially expressed metabolites,pseudouridine and3-Methoxy-4-Hydroxyphenylglycol Sulfate,within the key modules showed significant correlations with Reiff score(correlation coefficients of 0.40 and 0.32,respectively)and BSI score(correlation coefficients of0.41 and 0.39,respectively).Conclusion:1.PCD is the most common in hereditary bronchiectasis,with high heterogeneity and poor prognosis in some patients.2.In stable bronchiectasis patients,bacterial pathogens are the primary etiological agents,and there is a relatively high proportion of mixed bacterial infections.The presence of mixed bacterial infections in bronchiectasis patients is associated with poorer lung function.In diagnosing the microbiology of culture-negative bronchiectasis patients,metagenomic next-generation sequencing(m NGS)can be beneficial.3.The interaction network between the genera Neisseria and Moraxella and the host reflects the complex pathophysiological mechanisms underlying bronchiectasis.4.The serum differential metabolites of patients with bronchiectasis and healthy controls can be used as one of the markers of the severity of bronchiectasis,which provides a new idea for the study of the pathogenesis and therapeutic targets of bronchiectasis.