Molecular charaterization of Primary Ciliary Dyskinesia

Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2021-2022Introduction: Motile cilia are highly complex hair-like organelles protruding from the apical surface of epithelia cells of various human organ systems. They contain a 9+2 tubulin-based axoneme core structure and an important number of multiprotein complexes comprising the sub-structures: the dynein arms, the nexin links, the central sheath and the radial spokes. Primary ciliary dyskinesia (PCD) is an autosomal recessive rare disease (1-15,000 live-born children) caused by an alteration of ciliary structure and function, which impairs the clearance of respiratory secretions. Its clinical manifestations include chronic wet cough, secretory otitis media, rhinosinusitis, repeated episodes of bronchitis and/or recurrent pneumonia, bronchiectasis, male infertility, female subfertility, and situs inversus (50%) or heterotaxia (6%). PCD diagnosis is complex and based on a combination of techniques. Genetics and immunofluorescence have been recently proposed as reliable techniques to improve understanding of disease-causing genes and diagnosis rate in PCD. Objectives: Optimize genetic and immunofluorescence analysis to improve the diagnosis rate in our cohort and help understand the correlation between a specific genetic defect and ciliary structure and function. Methods: This was a multicenter cross-sectional study of patients with a high suspicion of PCD according to the European Respiratory Society criteria. To characterize the genetic alterations in our cohort, we designed a gene panel for massive sequencing that included 44 genes associated with PCD. Whole-exome sequencing was conducted on patients with a gene panel negative result. Immunofluorescence studies were carried out applying a four commercial fluorescently labeled antibody panel (DNAH5, DNALI1, GAS8 and RSPH4A or RSPH9) to study the presence and distribution of these ciliary proteins in nasal brushing respiratory cell samples. Results: Seventy-nine patients, 53 of whom had a diagnosis of confirmed or highly-likely PCD, were included in the genetic study. The sensitivity of the 44 PCD gene panel was 81.1%, with a specificity of 100%. Candidate variants were found in some of the genes of the panel in 43 patients with PCD, 51.2% (22/43) of whom were homozygotes and 48.8% (21/43) compound heterozygotes. The most common causative genes were DNAH5 and CCDC39. We found 52 different variants, 36 of which were not previously described in the literature and the most prevalent variant was detected in RSPH1 (c.85G>T/p.Glu39Ter). In nine patients with gene panel negative result and in one patient firstly considered unlikely PCD, whole-exome sequencing was carried out. Two patients presented candidate variants in two recently genes associated with PCD: CFAP300/C11orf70 and DNAAF6/PIH1D3. Four patients presented variants in candidate genes (GOLGA3 and C2CD3) which could explain their phenotype. Regarding immunofluorescence analysis, the four antibody-panel was tested in 74 nasal brushing samples. Sixty-eight (91.9%) patients were evaluable for all tested antibodies. Thirty-three cases (44.6%) presented an absence or mislocation of protein in ciliary axoneme (15 absent and 3 proximal distribution of DNAH5 in the ciliary axoneme, 3 absent DNAH5 and DNALI1, 7 absent DNALI1 and cytoplasmatic localization of GAS8, 1 absent GAS8, 3 absent RSPH9 and 1 absent RSPH4A). Fifteen patients had confirmed or highly-likely PCD but normal immunofluorescence results (68.8% sensitivity and 100% specificity), three of them with confirmed likely pathogenic variants in DNAH11. Considering our immunofluorescence results we proposed a two-step antibody panel: a first round with DNAH5, DNALI1, GAS8 and RSPH9 antibodies, and a second round, if required, with DNAH11 and SPEF2 antibodies. Conclusions: The design and implementation of our gene panel produces a high yield in the genetic diagnosis of PCD. Whole-exome sequencing analysis is a useful technique in PCD suspected cases with non-genetic variants in known PCD causing genes. Immunofluorescence analysis is a quick, available, low-cost and reliable diagnostic test for PCD, although it cannot be used as a standalone test