Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility.

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly.

Mutation of serine/threonine protein kinase 36 (STK36) causes primary ciliary dyskinesia with a central pair defect.

Primary ciliary dyskinesia (PCD) is a genetic condition of impaired ciliary beating, characterized by chronic infections of the upper and lower airways and progressive lung failure. Defects of the outer dynein arms are the most common cause of PCD. In about half of the affected individuals, PCD occurs with situs inversus (Kartagener syndrome). A minor PCD subgroup including defects of the radial spokes (RS) and central pair (CP) is hallmarked by the absence of laterality defects, subtle beating abnormalities, and unequivocally apparent ultrastructural defects of the ciliary axoneme, making their diagnosis challenging. We identified homozygous loss-of-function mutations in STK36 in one PCD-affected individual with situs solitus. Transmission electron microscopy analysis demonstrates that STK36 is required for cilia orientation in human respiratory epithelial cells, with a probable localization of STK36 between the RS and CP. STK36 screening can now be included for this rare and difficult to diagnose PCD subgroup.

Tyrosine kinase 2 is not limiting human antiviral type III interferon responses.

Tyrosine kinase 2 (TYK2) associates with interferon (IFN) alpha receptor, IL-10 receptor (IL-10R) beta and other cytokine receptor subunits for signal transduction, in response to various cytokines, including type-I and type-III IFNs, IL-6, IL-10, IL-12 and IL-23. Data on TYK2 dependence on cytokine responses and in vivo consequences of TYK2 deficiency are inconsistent. We investigated a TYK2 deficient patient, presenting with eczema, skin abscesses, respiratory infections and IgE levels >1000 U/mL, without viral or mycobacterial infections and a corresponding cellular model to analyze the role of TYK2 in type-III IFN mediated responses and NK-cell function. We established a novel simple diagnostic monocyte assay to show that the mutation completely abolishes the IFN-α mediated antiviral response. It also partly reduces IL-10 but not IL-6 mediated signaling associated with reduced IL-10Rß expression. However, we found almost normal type-III IFN signaling associated with minimal impairment of virus control in a TYK2 deficient human cell line. Contrary to observations in TYK2 deficient mice, NK-cell phenotype and function, including IL-12/IL-18 mediated responses, were normal in the patient. Thus, preserved type-III IFN responses and normal NK-cell function may contribute to antiviral protection in TYK2 deficiency leading to a surprisingly mild human phenotype.

TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization.

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex.

Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.

Immunofluorescence Analysis and Diagnosis of Primary Ciliary Dyskinesia with Radial Spoke Defects.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating, leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. Individuals with PCD caused by defective radial spoke (RS) heads are difficult to diagnose owing to lack of gross ultrastructural defects and absence of situs inversus. Thus far, most mutations identified in human radial spoke genes (RSPH) are loss-of-function mutations, and missense variants have been rarely described. We studied the consequences of different RSPH9, RSPH4A, and RSPH1 mutations on the assembly of the RS complex to improve diagnostics in PCD. We report 21 individuals with PCD (16 families) with biallelic mutations in RSPH9, RSPH4A, and RSPH1, including seven novel mutations comprising missense variants, and performed high-resolution immunofluorescence analysis of human respiratory cilia. Missense variants are frequent genetic defects in PCD with RS defects. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the RS head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1, fail to assemble RSPH9, whereas RSPH9 mutations result in axonemal absence of RSPH9, but do not affect the assembly of the other head proteins, RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants, or one amino acid deletion. Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants. RSPH4A is the core protein of the RS head.