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Summary Ciliopathies are a growing class of disorders caused by abnormal ciliary axonemal structure and function. Our understanding of the complex genetic and functional phenotypes of these conditions has rapidly progressed. Primary ciliary dyskinesia (PCD) remains the sole genetic disorder of motile cilia dysfunction. However, unlike many Mendelian genetic disorders, PCD is not caused by mutations in a single gene or locus, but rather, autosomal recessive mutation in one of many genes that lead to a similar phenotype. The first reported PCD mutations, more than a decade ago, identified genes encoding known structural components of the ciliary axoneme. In recent years, mutations in genes encoding novel cytoplasmic and regulatory proteins have been discovered. These findings have provided new insights into the functions of the motile cilia, and a better understanding of motile cilia disease. Advances in genetic tools will soon allow more precise genetic testing, mandating that clinicians must understand the genetic basis of PCD. Here, we review genetic mutations, their biological impact on cilia structure and function, and the implication of emerging genetic diagnostic tools.

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. We identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations.

H epatic fibrosis associated with inflammatory cell infiltration is a prominent feature of persistent infection by hepatitis B virus (HBV) and hepatitis C virus (HCV). The hepatic stellate cell (HSC) has assumed a central role in this response after its activation by inflammatory cytokines and mediators. [1][2][3][4][5] The cellmediated immune response after viral hepatitis reflects the activity of CD4ϩ helper T and CD8ϩ cytotoxic T

BackgroundPrimary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.MethodsGenetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.ResultsA single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.ConclusionMutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.

SARS-CoV-2 spike (S) variants govern transmissibility, responsiveness to vaccination and disease severity. In a screen for new models of SARS-CoV-2 infection, we identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2 expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Though the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Primary ciliary dyskinesia is a rare genetic disease of the motile cilia and is one of a rapidly expanding collection of disorders known as ciliopathies. Patients with primary ciliary dyskinesia have diverse clinical manifestations, including chronic upper and lower respiratory tract disease, left-right laterality defects, and infertility. In recent years, our understanding of the genetics of primary ciliary dyskinesia has rapidly advanced. A growing number of disease-associated genes and pathogenic mutations have been identified, which encode axonemal, cytoplasmic, and regulatory proteins involved in the assembly, structure, and function of motile cilia. Our knowledge of cilia genetics and the function of the proteins encoded has led to a greater understanding of the clinical manifestations of motile ciliopathies. These advances have changed our approach toward diagnostic testing for primary ciliary dyskinesia. In this review, we will describe how new insights into genetics have allowed us to define the clinical features of primary ciliary dyskinesia, revolutionize diagnostics, and reveal previously unrecognized genotype-phenotype relationships in primary ciliary dyskinesia.