Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of Primary Ciliary Dyskinesia. A. Onoufriadis1, A. Shoemark2, M. Munye3, C. James4, EM. Rosser5, C. Bacchelli4, SL. Hart3, M. Schmidts1, JE. Danke-Roelse6, G. Pals7, C. Hogg2, EMK. Chung8, . UK10K9, HM. Mitchison1 1) Molecular Medicine Unit and Birth Defects Research Centre, Institute of Child Health, University College London, London, UK; 2) Department of Paediatric Respiratory Medicine, Royal Brompton and Harefield NHS Trust, London, UK; 3) Molecular Immunology Unit, Institute of Child Health, University College London, London, UK; 4) Centre for Translational Genomics - GOSgene, Institute of Child Health, University College London, London, UK; 5) Clinical Genetics Unit, Great Ormond Street Hospital, London, UK; 6) Department of Pediatrics, Atrium Medical Center, Heerlen, the Netherlands; 7) Department of Clinical Genetics, VU University Medical Center, Amsterdam, the Netherlands; 8) General and Adolescent Paediatric Unit, Institute of Child Health, University College London, London, UK; 9)

   Primary ciliary dyskinesia (PCD) is a recessively inherited, genetically heterogeneous disorder arising from abnormal function of motile cilia and sperm flagella. It affects one per 15,000-30,000 births. Abnormal motility of cilia and flagella leads to symptoms including neonatal respiratory distress, chronic respiratory infections and obstructive lung disease, otitis media, subfertility and situs abnormalities. So far, mutations in 20 genes have been identified to cause PCD including DNAH5, DNAH11, DNAI1, DNAI2 and DNAL1 which encode subunits of the axonemal outer dynein arm (ODA) and CCDC114 which encodes an ODA docking complex component. Exome-sequencing was performed in one PCD patient at the Wellcome Trust Sanger Institute (Cambridge, UK) as part of the UK10K project. This affected individual is from a consanguineous marriage so we focused on homozygous non-synonymous or splice-site substitutions or indels, that were novel or present in the 1000 Genomes Project with a frequency 0.01. To prioritize candidate genes we used our internal allele count data, removing variants detected more than ten times across the UK10K cohort, as PCD causing mutations would not likely appear in multiple well-phenotyped non-PCD patients. This filtering strategy revealed a homozygous protein-truncating nonsense variant in ARMC4 (c.2675CA; pSer892*), a gene previously shown to be involved in ciliogenesis. In parallel, whole genome sequencing was performed in the two parents of a consanguineous marriage since material from their affected offspring was not available. We filtered per chromosome, to identify protein altering heterozygous variants shared by both parents with a 1000 Genomes MAF0.01. This strategy revealed a second ARMC4 protein-truncating nonsense variant (c.1972GT; p.Glu658*). Segregation analysis in all available members of the pedigrees confirmed recessive inheritance of both c.2675CA and c.1972GT variants. Respiratory cilia from affected individuals were immotile and ultrastructural analysis by transmission electron microscopy showed loss of the ciliary outer dynein arms. ARMC4 transcript levels were investigated by means of qPCR in respiratory epithelial cells during ciliogenesis, showing a highly significant increase of ARMC4 expression in ciliated cells when compared to non-ciliated basal cells. Together these data suggest that ARMC4 mutations cause PCD due to loss of the outer dynein arms.

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