Mutation Screening And Gene Diagnosis Of Human Primary Ciliary Dyskinesia

Background:Primary cilia dyskinesia（PCD）is a rare clinical syndrome that can affect multiple organs and tissues.The prevalence in the population is about 1/10000-1/20000.Since the widespread distribution of cilia,its clinical symptoms are highly heterogeneous,including recurrent pulmonary infections,bronchiectasis,chronic sinusitis,otitis media,visceral organ displacement and male infertility,etc.PCD patients usually present with respiratory symptoms as the initial manifestation.Due to the diversity of symptoms and signs of PCD patients,there is still no unified diagnostic criteria for PCD.In recent years,the genetic etiology and molecular regulatory mechanism of PCD have been studied and understood more deeply,the genetic etiology and gene diagnosis of PCD have been paid more attention.Purpose:Whole exome sequencing（WES）analysis was performed in 10 patients clinically highly suspected of PCD to screen the relevant variants of the known PCD pathogenic genes firstly,and then to screen for new candidate pathogenic genes of PCD in cases without clear known pathogenic genes.We aim to providetheoretical basis for the genetic etiology and gene diagnosis of PCD,as well as genetic counseling and individualized treatment for patients with PCD.Method:Ten clinical cases highly suspected of PCD were collected from Department of Respiratory and Critical Care Medicine,the Second Affiliated Hospital of Anhui Medical University and Reproductive Medicine Center,the First Affiliated Hospital of Anhui Medical University（7 cases were from consanguineous families）,numbered as PCD 1-PCD 10.Peripheral blood was sampled with informed consent for WES sequencing.Subsequentl data filtering,variant screening and bioinformatics analysis were performed.Firstly,known PCD pathogenic genes and inheritance modes were collected through literature and database research,and the screened variants were compared to identify the pathogenic variants in the patients.Secondly,in the cases where the known pathogenic genes could not explain,combined with the family history of the patients,the focus was on the genes with low frequency,cilia-specific expression or high abundance,and the specific function of the genes,to screen out the new candidate pathogenic genes for PCD.Thirdly,Sanger sequencing was used to verify the known pathogenic variants and candidate pathogenic variants screened out,and family cosegregation analysis was used to determine the source of the variants.Finally,considering the invasiveness of cilia sampling from the respiratory tract and the similarity between cilia and sperm flagella,the ultrastructure changes of sperm flagella were detected by transmission electron microscopy in male patients’ sperm samples,and the quantitative and positional expression changes of the genes were detected by H&E staining,immunofluorescence technology,real-time fluorescence PCR and Western Blot technology,etc.Result:After strict variant screening and comparison with known PCD pathogenic genes,we identified 13 deleterious variants in 7 known pathogenic genes from 9 patients,including CCDC39（2 cases）,LRRC6（2 cases）,DNAI1（1 case）,DNAH5（1 case）,DNAH11（1 case）,HYDIN（1 case）,and NME5（1 case）,involving multiple PCD pathogenesis mechanisms such as dynein arm structural protein deficiency,dynein arm cytoplasmic preassembly related protein deficiency,and centriolar related protein deficiency.（1）Four dynein arm structure and preassembly related genes:①Patients PCD2 and PCD3,who are siblings,both carry homozygous frameshift mutation c.64dupT:p.S22fs of LRRC6 gene;②Patient PCD6 carries homozygous stopgain mutation c.9050G>A;p.W3017X of DNAH11 gene,with sperm showing typical multiple morphological abnormalities and ultrastructural deficiency in the sperm flagella;③Patient PCD7 carries homozygous stopgain mutation c.C610T:p.Q204X in DNAI1 gene;④Patient PCD9 carries compound heterozygous variants c.7915C>T:p.R2639X/c.4703G>A:p.R1568H in DNAH5 gene.（2）Other three PCD related genes:①Patient PCD1 and PCD4 carry compound heterozygous variants of CCDC39 gene,c.286C>T:p.R96X and c.732₇33delCA:p.C244fs/c.2802₂803insGTT,p.K935delinsVK,respectively.The sperm of PCD4 showed typical multiple multiple morphological abnormalities in the sperm flagella,and the western blot result showed significantly decreased expression of CCDC39 protein in the sperm.②Patient PCD5 carries compound heterozygous variants of HYDIN gene,c.6316+1G>A/c.5969-2A>G,which also led to multiple morphological abnormalities and ultrastructural deficiency in the sperm flagella of the patient.Besides,the expression of HYDIN and its related proteins SPEF2,SPAG6,RSPH1 and RSPH3 were significantly reduced in the patient’s sperm.③Patient PCD8 carries compound heterozygous variants of NME5 gene,c.T209A:p.L70X/c.163delA:p.S55Vfs.In addition,we identified a novel PCD candidate gene ARMC2.In patient PCD 10,we identified a homozygous stopgain mutation of ARMC2 gene,c.C1264T:p.R422X.Through H&E staining,we found that the sperm of patient PCD 10 showed typical multiple morphological abnormalities in the sperm flagella,mainly short and curved tail.Through transmission electron microscopy,we observed that the homozygous mutation of ARMC2 gene caused disorganization of the "9+2" structure of microtubules in the patient’s sperm,including the lack of some central and peripheral microtubules.Conclusion:Monogenic mutation is the main cause of PCD.Whole exome sequencing technology not only can help us to to accurately identify the pathogenic gene of PCD,but also provides the basis for PCD gene diagnosis and enhances the reliability of clinical diagnosis.In the 10 clinically highly suspected PCD patients we collected,9 patients can be explained by known PCD pathogenic genes.In addition,our research results also suggested that ARMC2 may be a new candidate gene of PCD in an autosomal recessive inheritance pattern,of which homozygous or compound heterozygous variants may lead to the occurrence of PCD.Our findings provide some new theoretical basis for the genetic etiology,gene diagnosis and genetic counseling of PCD,and provide certain reference for the individualized treatment of such patients with PCD.