Identification Of The Disease-causing Gene Variants In A Family With Primary Ciliary Dyskinesia

Background: Primary ciliary dyskinesia(PCD)is a rare genetic disease,caused by structural and/or functional defects of cilia,predominantly inherited in an autosomal recessive fashion.The clinical manifestations of PCD patients are diverse,mainly including chronic rhinitis,chronic sinusitis,chronic otitis media,recurrent respiratory tract infection,bronchiectasis,and infertility.In addition,about 50% of PCD patients display situs inversus,known as Kartagener syndrome.At present,over 40 PCD-associated genes have been identified.Objective: This study used the strategy of whole exome sequencing combined with Sanger sequencing to identify the disease-causing gene variants in a family with PCD.Methods: A three-generation Han-Chinese family with PCD was recruited from the Third Xiangya Hospital,Central South University.Five members of this family participated in this study,clinical data and peripheral venous blood samples were collected and relevant clinical examinations were conducted on them.Genomic DNA was extracted in accordance with a normative phenol-chloroform extraction method.Whole exome sequencing was conducted in the proband genomic DNA sample.The sequencing data were compared with the National Heart,Lung,and Blood Institute Exome Sequencing Project 6500,1000 Genomes Project,the Single Nucleotide Polymorphism database(version141),Genome Aggregation Database and the Exome Aggregation Consortium database,and the BGI in-house exome database to screen for candidate pathogenic variants.Sanger sequencing was conducted in family members’ g DNA samples to analyse the candidate pathogenic variants.Phenotype-genotype co-segregation analysis within this family was carried out to further clarify the candidate pathogenic variants.Bioinformatic prediction softwares were used to perform functional prediction and conservation analysis of the candidate variants.In vitro minigene splicing assay was applied to evaluate the impact of the splicing variant on the splicing process.The classification of the candidate variants was performed according to the 2015 American College of Medical Genetics and Genomics(ACMG)revised interpretation guidelines for variants.Results: Compound heterozygous variants,c.1974-1G>C and c.7787G>A [p.(Arg2596Gln)],in the dynein axonemal heavy chain 11gene(DNAH11)were identified as candidate disease-associated variants of this PCD patient.Sanger sequencing verified the proband carried compound heterozygous DNAH11 variants(c.1974-1G>C and c.7787G>A),and his unaffected daughter and son carried heterozygous DNAH11 c.1974-1G>C and c.7787G>A variants,respectively,indicating the compound heterozygous variants co-segregated with the family’s PCD phenotype.Bioinformatic prediction softwares predicted the splicing variant c.1974-1G>C may destroy the acceptor site,and missense variant c.7787G>A [p.(Arg2596Gln)] was deleteriousis.Conservative analysis results revealed the DNAH11 protein arginine residue at site2596 was highly conserved in many species.The results of minigene assay found the c.1974-1G>C variant could cause skipping over exon 12,predicted to result in a truncated protein.Based on the ACMG guidelines,the DNAH11 c.1974-1G>C and c.7787G>A variants were categorized as“pathogenic” and “likely pathogenic”,respectively.Conclusions: This study identified compound heterozygous DNAH11 variants,c.1974-1G>C and c.7787G>A,may be the genetic cause for a Han-Chinese PCD family.This study may broaden the spectrum of PCD variants and facilitate pinpoint diagnosis and prognosis.Figures 9,Tables 28,References 50...