HYDIN variants cause primary ciliary dyskinesia in the Finnish population.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by chronic respiratory tract infections and in some cases laterality defects and infertility. The symptoms of PCD are caused by malfunction of motile cilia, hair-like organelles protruding out of the cell. Thus far, disease causing variants in over 50 genes have been identified and these variants explain around 70% of all known cases. Population specific genetics underlying PCD has been reported highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics and management. METHODS: Whole exome sequencing was used to identify disease causing variants in Finnish PCD cohort. The effect of the identified HYDIN variants on cilia structure and function was confirmed by high-speed video analysis, immunofluorescence and electron tomography. RESULTS: In this study, we identified three Finnish PCD patients carrying homozygous loss-of-function variants and one patient with compound heterozygous variants within HYDIN. The functional studies showed defects in the axonemal central pair complex. All patients had clinical PCD symptoms including chronic wet cough and recurrent airway infections, associated with mostly static airway cilia. CONCLUSION: Our results are consistent with the previously identified important role of HYDIN in the axonemal central pair complex and improve specific diagnostics of PCD in different national populations.

Are peanut oral food challenges still useful? An evaluation of children with suspected peanut allergy, sensitization to Ara h 2 and controlled asthma.

BACKGROUND: Sensitization to Ara h 2 has been proposed as a promising biological marker for the severity of peanut allergy and may reduce the need for oral food challenges. This study aimed to evaluate whether peanut oral food challenge is still a useful diagnostic tool for children with suspected peanut allergy and an elevated level of Ara h 2-specific IgE. Additionally, we assessed whether well-controlled asthma is an additional risk for severe reactions. METHODS: A retrospective analysis of 107 children with sensitization to Ara h 2-specific IgE (> 0.35 kU/l) undergoing open peanut challenges during 2012-2018 in the Tampere University Hospital Allergy Centre, Finland. RESULTS: Of the 107 challenges, 82 (77%) were positive. Serum levels of Ara h 2 -sIgE were higher in subjects with a positive challenge than in those who remained negative (median 32.9 (IQR 6.7-99.8) vs. 2.1 (IQR 1.0-4.9) kU/l), p < 0.001) but were not significantly different between subjects with and without anaphylaxis. No correlation was observed between the serum level of Ara h 2-sIgE and reaction severity grading. Well-controlled asthma did not affect the challenge outcome. CONCLUSIONS: Elevated levels of Ara h 2-specific IgE are associated with a positive outcome in peanut challenges but not a reliable predictor of reaction severity. Additionally, well-controlled asthma is not a risk factor for severe reactions in peanut challenges in children with sensitization to Ara h 2.

CFAP300 mutation causing primary ciliary dyskinesia in Finland.

Primary ciliary dyskinesia (PCD) is a rare genetic condition characterized by chronic respiratory tract infections and in some cases laterality defects and infertility. The symptoms of PCD are caused by malfunction of motile cilia, hair-like organelles protruding out of the cell that are responsible for removal of mucus from the airways and organizing internal organ positioning during embryonic development. PCD is caused by mutations in genes coding for structural or assembly proteins in motile cilia. Thus far mutations in over 50 genes have been identified and these variants explain around 70% of all known cases. Population specific genetics underlying PCD has been reported, thus highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics. In this study, we identified a recurrent loss-of-function mutation c.198_200delinsCC in CFAP300 causing lack of the protein product. PCD patients homozygous for the identified CFAP300 mutation have immotile airway epithelial cilia associated with missing dynein arms in their ciliary axonemes. Furthermore, using super resolution microscopy we demonstrate that CFAP300 is transported along cilia in normal human airway epithelial cells suggesting a role for CFAP300 in dynein complex transport in addition to preassembly in the cytoplasm. Our results highlight the importance of CFAP300 in dynein arm assembly and improve diagnostics of PCD in Finland.

High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a congenital disorder predominantly inherited in an autosomal recessive trait. The disorder causes disturbance in the motion of cilia, leading to severe impairment of mucociliary clearance (MCC). If undiagnosed or diagnosed too late, the condition leads to the development of bronchiectasis and serious damage to the lungs in later life. Most of the methods for diagnosing PCD are time-consuming and demand extensive economic resources to establish them. High-speed video microscopy analysis (HSVMA) is the only diagnostic tool to visualize and analyze living respiratory cells with beating cilia in vitro. It is fast, cost-effective, and, in experienced hands, very reliable as a diagnostic tool for PCD. In addition, classical diagnostic measures such as transmission electron microscopy (TEM) are not applicable for some mutations as morphological changes are absent. This paper describes the process of collecting respiratory epithelial cells, the further preparation of the specimen, and the process of HSVMA. We also describe how brushed cells can be successfully kept unharmed and beating by keeping them in a nourishing medium for storage and transport to the investigation site in cases where a clinic does not possess the equipment to perform HSVMA. Also shown are videos with pathologic beating patterns from patients with a mutation in the dynein arm heavy chain 11 gene (DNAH11), which cannot be diagnosed with TEM; the result of an inconclusive HSVMA due to infection of the upper airways, as well as an unsuccessful brushing with superimposition of red blood cells. With this article, we would like to encourage every unit dealing with pulmonology patients and rare lung diseases to perform HSVMA as part of their daily routine diagnostics for PCD or send the specimens over to a center specializing in performing HSVMA.

Two novel mutations in the DNAH11 gene in primary ciliary dyskinesia (CILD7) with considerable variety in the clinical and beating cilia phenotype.

BACKGROUND: Diagnosis of primary ciliary dyskinesia (PCD) still remains a challenge, especially with mutations in the Dynein Arm Heavy Chain 11 (DNAH11) gene. Classical diagnostic measures like Transmission Electron Microscopy (TEM) are not applicable for mutations in the DNAH11 gene since ultrastructural defects of the ciliary apparatus are absent. Novel mutations encoding for PCD appear all the time with considerable variation in the clinical picture, making it necessary to update data bases and guidelines for PCD diagnostics. METHODS: In this study we examined two unrelated, Finnish families with symptoms of PCD applying the clinical scoring system: Primary ciliary dyskinesia Rule (PICADAR), high speed video microscopy analysis (HSVMA) for ciliary movement, a commercially available gene panel analysis and nasal Nitric Oxide (nNO) measurements if applicable. RESULTS: Two, likely pathogenic variants in the DNAH11 gene (c.2341G > A, p. (Glu781Lys) ja c.7645 + 5G > A) were detected. In the first family, compound heterozygous mutations led to disease manifestation in two of 4 children, which showed a similar phenotype of cilia beating pattern but marked differences in disease severity. In the second family, all three children were homozygotes for the c.2341G > A p.(Glu781Lys) mutation and showed a similar degree of disease severity. However, the phenotype of cilia beating pattern was different ranging from stiff, static cilia to a hyperkinetic movement in one of these children. CONCLUSIONS: In this study we describe two Finnish families with PCD, revealing two novel mutations in the DNAH11 gene which show considerable variety in the clinical and beating cilia phenotype. The results of this study show the clinician that PCD can be much milder than generally expected and diagnosis demands a combination of measures which are only successful in experienced hands. Chronic and repeatedly treated wet cough should raise suspicion of PCD, referring the patient for further diagnostics to a specialised PCD centre.

Onset of action of inhaled glucocorticoids on bronchial and alveolar nitric oxide output.

Fractional exhaled nitric oxide (FENO) is a marker of airway inflammation. Measuring FENO at multiple flow rates enables calculation of NO parameters: bronchial NO output (J awNO), bronchial wall (C awNO) and alveolar (C ANO) NO concentrations, and bronchial diffusion factor of NO (D awNO). FENO is known to rapidly reduce after the commencement of inhaled corticosteroid (ICS) treatment. However, little is known on the effect of ICS on the other NO parameters. We assessed (1) the onset of action of ICS treatment on the NO parameters and (2) whether the changes in bronchial NO output are due to changes in bronchial wall NO concentration or diffusion factor. FENO and other NO parameters were measured at baseline and after 1, 3 and 7 d of treatment with inhaled fluticasone propionate 250 µg b.i.d. in 23 allergic children with a history of asthma-like symptoms. There was a decrease in J awNO (from 680 (244/1791) (median (1st/3rd quartile)) to 357 (165/753) pl s-1, p < 0.001) and FENO50( from 13.8 (7.5/35) to 8.3 (5.36/17.0) ppb, p < 0.001) in 3 d from the first dose of ICS. Also, C awNO seemed to reduce after 3 d (from 171 (89/328) to 79 (54/157) ppb, p = 0.041), while D awNO remained unchanged. Furthermore, C ANO reduced during the 7 d treatment (from 3.0 (2.0/5.0) to 2.3 (1.9/2.6) ppb, p = 0.004). ICS treatment reduced FENO50 and J awNO rapidly and the decline was caused by decreased bronchial wall NO concentration while bronchial NO diffusion factor remained unchanged. These findings suggest that C awNO could be a more specific marker of airway inflammation and treatment response than J awNO or FENO50, which are both determined also by D awNO that seems to be resistant to the treatment with ICS.

Wheat oral immunotherapy was moderately successful but was associated with very frequent adverse events in children aged 6-18 years.

AIM: This study investigated oral immunotherapy (OIT) for children aged 6-18 years with wheat allergies. METHODS: Well-cooked wheat spaghetti was given to 100 children with wheat allergies every day for 17 weeks, increasing from 0.3 to 2000 mg of wheat protein, followed by three- and nine-month maintenance phases. Blood samples were taken before therapy and at follow-up visits. The study was carried out in 2009-2015 in four Finnish paediatric allergology units. RESULTS: The children (67% male) had a mean age of 11.6 years (range 6.1-18.6), and 57 were using wheat daily 16 months after the initiation of therapy. Allergic symptoms occurred in 94/100 children: mild in 34, moderate in 36 and severe in 24. Specific immunoglobulin E (IgE) for ω-5-gliadin was significantly higher in patients who did not reach the target dose and were related to the intensity of reactions. CONCLUSION: The majority (57%) of children with wheat allergies could use wheat in their daily diet 16 months after the initiation of OIT, but 94/100 had adverse reactions and 60 were moderate or severe. Specific IgE to ω-5-gliadin may provide a biomarker for how much wheat can be tolerated and the intensity of the reactions to immunotherapy.

Expression of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator messenger ribonucleic acids and proteins in rat and human testis.

Dioxins, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), use the aryl hydrocarbon receptor (AHR)/aryl hydrocarbon receptor nuclear translocator (ARNT) receptor complex to mediate their toxic actions. In addition to interaction with environmental pollutants, several transcription factors, steroid receptors, and growth factors are capable interacting with the AHR/ARNT complex, which suggests a constitutive role for the receptor complex. The testis has been reported to be among the most sensitive organs to TCDD exposure. Our experiments revealed a complex distribution of AHR and ARNT mRNAs and proteins in rat and human testis. AHR and ARNT immunoreactivities could be detected in the nuclei of interstitial and tubular cells. The incubation of seminiferous tubules in a serum-free culture medium resulted in up-regulation of AHR mRNA, which could be depressed by adding FSH to the culture medium. Furthermore, the incubation of tubular segments with a solution of 1 or 100 nM TCDD resulted in a 2- to 3-fold increase in apoptotic cells. Thus, up-regulation of AHR in cultured tubular segments and consecutive depression by FSH suggest a role for AHR in controlled cell death during spermatogenesis. We suggest that AHR and ARNT mediate effects by direct action on testicular cells in the rat and human testis.