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.

Reduced generation of multiple motile cilia (RGMC) is a novel chronic destructive airway disease within the group of mucociliary clearance disorders with only few cases reported. Mutations in two genes, CCNO and MCIDAS, have been identified as a cause of this disease, both leading to a greatly reduced number of cilia and causing impaired mucociliary clearance. This study was designed to identify the prevalence of CCNO mutations in Israel and further delineate the clinical characteristics of RGMC. We analyzed 170 families with mucociliary clearance disorders originating from Israel for mutations in CCNO and identified two novel mutations (c.165delC, p.Gly56Alafs*38; c.638T>C, p.Leu213Pro) and two known mutations in 15 individuals from 10 families (6% prevalence). Pathogenicity of the missense mutation (c.638T>C, p.Leu213Pro) was demonstrated by functional analyses in Xenopus. Combining these 15 patients with the previously reported CCNO case reports revealed rapid deterioration in lung function, an increased prevalence of hydrocephalus (10%) as well as increased female infertility (22%). Consistent with these findings, we demonstrate that CCNO expression is present in murine ependyma and fallopian tubes. CCNO is mutated more frequently than expected from the rare previous clinical case reports, leads to severe clinical manifestations, and should therefore be considered an important differential diagnosis of mucociliary clearance disorders.

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.

[537][538][539][540][541][542][543][544][545][546][547][548] 2002]. We now show that EF increases fluid absorption across monolayers of rat FDLE in a dose-dependent manner. To study the role of subunits of the epithelial Na ϩ channel (ENaC) in the phenomena, we cultured explants from the distal lungs of 16-day gestational age wild-type (WT) or ␣-, ␤-, or ␥-ENaC knockout or heterozygote (HT) mice. WT explants cultured in media continuously expanded over time as a result of net fluid secretion. In contrast, when explants were exposed to EF for 24 h, net fluid absorption occurred. EF-exposed explants had normal histology, but marked changes were seen after Triton X-100 or staurosporine exposure. Transmission electron microscopy showed EF promoted lamellar body formation and abundant surfactant in the explants' lumens. EF-induced changes in explant size were similar in ␣-ENaC knockout, WT, and HT littermate fetal lung explants (P Ͼ 0.05). In contrast, EF's effect was attenuated in ␤-and ␥-ENaC knockouts (P Ͻ 0.05) vs. WT and HT littermate fetal lung explants. EF exposure slightly decreased or had no effect on mRNA levels for ␣-ENaC in various mouse genotypes but decreased expression of ␤-and ␥-ENaC subunit mRNAs (P Ͻ 0.01) across all genotype groups. We conclude that ␤-and ␥-, but not ␣-, ENaC subunits are essential for EF to exert its maximal effect on net fluid absorption by distal lung epithelia. epithelium; Na ϩ transport; lung development; amiloride-sensitive epithelial Na ϩ channel PULMONARY EDEMA can mainly occur as a result of one or both of the following mechanisms: increased transvascular pressure in the pulmonary microvasculature, as occurs in congestive heart failure, or increased permeability of the alveolar-capillary membrane to solutes, as occurs in adult respiratory distress syndrome. The ability of the lungs to clear this air space fluid has important clinical implications. For example, others have demonstrated that the active absorption of air space fluid in adults with pulmonary edema correlates with improved survival (20). Air space fluid clearance is actively driven by epithelial Na ϩ transport, which crosses the apical membrane and is then extruded across the basolateral membrane by Na ATPase (18, 19). The rate-limiting step in active Na ϩ transport by epithelia is the number and activity of the apical membrane Na ϩ channels. One of these Na ϩ channels is the amiloridesensitive epithelial Na ϩ channel (ENaC) that is composed of ␣-, ␤-, and ␥-subunits (6). However, it has been shown that normal mammalian lung epithelia have both an amiloridesensitive and an amiloride-insensitive component to their active Na ϩ transport both in vivo (24) and in vitro (8, 25). Presently, the molecular basis of this amiloride-insensitive Na ϩ transport is poorly understood. Regardless, if one could determine mechanisms underlying the regulation of this amiloride-sensitive and -insensitive Na ϩ transport, one could in the future identify novel approaches for the therapy of patients with pulmonary edema.It has been...

AimWe evaluated the use of computerized quantification of wheezing and crackles compared to a clinical score in assessing the effect of inhaled albuterol or inhaled epinephrine in infants with RSV bronchiolitis.MethodsComputerized lung sounds analysis with quantification of wheezing and crackles and a clinical score were used during a double blind, randomized, controlled nebulized treatment pilot study. Infants were randomized to receive a single dose of 1 mgr nebulized l-epinephrine or 2.5 mgr nebulized albuterol. Computerized quantification of wheezing and crackles (PulmoTrack®) and a clinical score were performed prior to, 10 minutes post and 30 minutes post treatment. Results were analyzed with Student's t-test for independent samples, Mann-Whitney U test and Wilcoxon test.Results15 children received albuterol, 12 received epinephrine. The groups were identical at baseline. Satisfactory lung sounds recording and analysis was achieved in all subjects. There was no significant change in objective quantification of wheezes and crackles or in the total clinical scores either within the groups or between the groups. There was also no difference in oxygen saturation and respiratory distress.ConclusionComputerized lung sound analysis is feasible in young infants with RSV bronchiolitis and provides a non-invasive, quantitative measure of wheezing and crackles in these infants. Trial registration number: ClinicalTrials.gov NCT00361452

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Our results suggest that EGG abnormalities are frequently found in CF patients. The similar rate of EGG and gastric scintigraphy abnormalities suggests that EGG may be a useful clinical tool in CF patients.

At birth, lung fluid produced during fetal life must be cleared immediately and efficiently before the first breath takes place, in order for infants to achieve a normal and successful transition from prenatal to postnatal life. Postnatal lung fluid resorption is mediated through activation of airway epithelial sodium channels (ENaC). The observation that ENaC expression is a gestational age-dependent process contributes to our understanding of the development of respiratory distress in both term and preterm infants due to impaired clearing of fluid from their lungs. As fluid absorption, mediated by ENaC activity, in postnatal life has a significant biological role in preventing respiratory distress, any strategy that enhances ENaC activity can potentially help to decrease its incidence and associated morbidity.