You are here

  • Home
  • Archive
  • Volume 79, Issue 11
  • Impact of TAS2R38 polymorphisms on nasal nitric oxide and Pseudomonas infections in primary ciliary dyskinesia: relation to genotype

Article Text

Article menu
Respiratory infection
Original research
Impact of TAS2R38 polymorphisms on nasal nitric oxide and Pseudomonas infections in primary ciliary dyskinesia: relation to genotype
  1. Massimo Pifferi1,
  2. Attilio Boner2,
  3. Debora Maj1,
  4. Angela Michelucci3,
  5. Gabriele Donzelli4,
  6. Angela M Cangiotti5,
  7. Raffaella Guazzo6,
  8. Giulia Bertolucci1,
  9. Veronica Bertini7,
  10. Chiara Doccioli8,
  11. Michele Piazza2,
  12. Angelo Valetto7,
  13. Maria Adelaide Caligo3,
  14. Diego Peroni1,
  15. Andrew Bush9,10
  1. 1 Department of Pediatrics, Pisa University Hospital, Pisa, Italy
  2. 2 Department of Surgical Science, Dentistry, Gynecology and Pediatrics, Integrated University Hospital of Verona, Verona, Italy
  3. 3 Laboratory of Molecular Genetics, Pisa University Hospital, Pisa, Italy
  4. 4 Institute of Clinical Physiology National Research Council, Pisa, Italy
  5. 5 Institute of Normal Human Morphology, Electron Microscopy Unit, University Hospital of Ancona Umberto I G M Lancisi G Salesi, Ancona, Italy
  6. 6 Laboratory of Electron Microscopy, Pathology Unit, Siena University Hospital, Siena, Italy
  7. 7 Laboratory of Cytogenetics, Pisa University Hospital, Pisa, Italy
  8. 8 Department of Statistics, Computer Science and Applications, University of Florence, Florence, Italy
  9. 9 Imperial College London, London, UK
  10. 10 Royal Brompton Hospital, London, UK
  1. Correspondence to Dr Massimo Pifferi; m.pifferi{at}med.unipi.it

Abstract

Objective Primary ciliary dyskinesia (PCD) severity has been related to genotype and levels of nasal nitric oxide (nNO). The most common TAS2R38 haplotypes (PAV/PAV, PAV/AVI, AVI/AVI) encoding the bitter taste receptor can affect nNO levels and thus could play a role in the susceptibility to respiratory infections. We assessed the impact of these polymorphisms on nNO production and Pseudomonas aeruginosa (P.a.) infections in different PCD genotypes.

Methods Prospective, longitudinal, single-centre study in patients with PCD with known genotype and one of three TAS2R38 haplotypes evaluated for up to 10 years. We related nNO values to TAS2R38 haplotypes in all patients, and in the three most frequent genotypes (CCDC39/CCDC40, DNAH5, DNAH11). In the genetic group(s) with different mean trends of nNO in relation to the polymorphism, we evaluated longitudinal lung function as a clinical outcome measure. We also studied any associations between the prevalence of chronic P.a. infection and PAV alleles. Linear mixed-effects models were used to evaluate longitudinal associations.

Results 119 patients with PCD underwent 1116 study visits. Only in the DNAH11 mutations group was there a mean trend of nNO production which was significantly higher in PAV/PAV than AVI/AVI haplotype (p=0.033), with a better trend in spirometric and plethysmographic parameters. In patients with DNAH11 mutations the PAV allele was also associated with a significantly reduced prevalence of chronic P.a. infection.

Conclusion TAS2R38 may be a modifier gene for PCD severity, but only in mild phenotype disease. Further study of TAS2R38 polymorphisms might enable new management strategies to prevent chronic P.a. infections.

  • Primary ciliary dyskinesia
  • Rare lung diseases
  • Bacterial Infection

Data availability statement

Data are available upon reasonable request. De-identified participant data are available from the corresponding author upon reasonable request, subject to the terms of Ethics Committee approval.

https://doi.org/10.1136/thorax-2024-221396

Statistics from Altmetric.com

    Request Permissions

    If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • The severity of lung disease in primary ciliary dyskinesia (PCD) has been related to genotype and nasal nitric oxide (nNO) production. The most common TAS2R38 haplotypes (PAV/PAV, PAV/AVI, AVI/AVI) encoding the bitter taste receptor can affect nNO levels and could play a role in the susceptibility to respiratory infections by modulating the NO pathway.

    WHAT THIS STUDY ADDS

    • nNO production was significantly higher in PAV/PAV compared with AVI/AVI haplotype but only in patients with DNAH11 mutations. The PAV/PAV polymorphism was associated with a more favourable trend in spirometric and plethysmographic parameters. It was also associated in the same group with a significantly reduced prevalence of chronic Pseudomonas aeruginosa (P.a.) infection. Thus, TAS2R38 may be a modifier gene for PCD severity, but only in mild phenotype disease.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • Further study of TAS2R38 polymorphisms might help to improve management strategies to prevent chronic P.a. infections.

    Introduction

    Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder, characterised by dysfunction of motile cilia,1 resulting in recurrent or chronic bacterial infections of the airways. The considerable variability in the severity and progression of lung disease has been related to the underlying PCD causing genes,2–6 as well as to airway nitric oxide (NO) concentrations.7 NO protect against infection, but it is reduced in the overwhelming majority of patients with PCD. Thus, low nasal NO (nNO) values are used in the diagnostic work up of PCD in the setting of an appropriate clinical phenotype.8 However, some but not all affected individuals may have unequivocally normal nNO levels,9 10 particularly if they have causative mutations in CCDC103 and RSPH1, or in RPGR, GAS8, CCNO, CFAP221, DNAH9, FOXJ1, GAS2L2, LRRC56, NEK10, SPEF2, STK36 and TTC12.11 12 Normal nNO values were also found in at least one patient with biallelic mutations in SPAG1, DNAAF1, DYX1C1, ZMYND10, CCDC40, RSPH4A, RSPH9, DNAJB13, DNAH5, TTC25, DNAH11 and HYDIN.7 13 Moreover, in patients with PCD with biallelic mutations in CCDC39 and CCDC40, the group known to have a more severe clinical course, (compared with DNAH5 and DNAH11) nNO decreased significantly over time and there was an associated higher prevalence of Pseudomonas aeruginosa (P.a.) infections.7

    The bitter taste receptor T2R38 is expressed in motile respiratory cilia. It is activated by bacterial products secreted by P.a. and other gram-negative bacteria, resulting in calcium-dependent generation of bactericidal quantities of NO.14–16 The TAS2R38 gene which encodes T2R38 has three common haplotypes, one homozygous for a functional receptor (PAV/PAV), another homozygous non-functional receptor (AVI/AVI) and a heterozygous haplotype (PAV/AVI) with intermediate function.17–20 Thus, TAS2R38 polymorphisms could affect nNO levels independent of PCD genotype,15 and for at least some patients, this could play a role in the susceptibility to respiratory infections.14 Interestingly, a lower prevalence of P.a. infection was found in a small series of patients with PCD with the TAS2R38 PAV/PAV haplotype.21 However, in cystic fibrosis, the PAV allele was reported to have no effect on,22 or be associated with a reduction in chronic P.a. infection.23

    In summary, there is variability in nNO levels within and between PCD genotypes. TAS2R38 may be activated by bacterial products with associated rapid NO production that can directly kill P.a. There is the variable prevalence of chronic P.a. infection in patients with PCD. Finally, some TAS2R38 polymorphisms may be associated with increased NO production thus ameliorating infection.

    Thus, we hypothesised that TAS2R38 polymorphisms affect NO levels in patients with PCD. We investigated the mean trends over time of nNO measurements in the haplotypes PAV/PAV, PAV/AVI and AVI/AVI in all our patients with PCD irrespective of genotype. We also compared the effects of TAS2R38 polymorphisms in the three most frequent genetic groups (CCDC39/CCDC40, DNAH5 and DNAH11). Furthermore, we evaluated lung function over time as a clinical outcome measure for each TAS2R38 haplotype in all of the above group(s) related to significant differences in mean trends of nNO production.

    The secondary aim was to estimate the overall age-related occurrence of P.a. infection and relate chronic infection lower airway with P.a. to the PAV allele. We also determined the number of children infected with P.a. and their age of first infection in relation to the presence of one or two PAV alleles.

    Material and methods

    Study design

    This was a longitudinal, single-centre, observational study.

    Study population and inclusion criteria

    This study included patients with adult and paediatric PCD, attending the Department of Paediatrics University Hospital of Pisa as outpatients. All had a confirmed diagnosis of PCD.24 We enrolled only patients with PCD with known biallelic mutations in PCD-causing genes determined by next-generation sequencing for 50 genes and by determining the carrier status in the parents; or by a customised array comparative genomic hybridisation if we had identified a single or no pathogenetic mutation. Enrolled patients were required also to have one of the TAS2R38 haplotypes of the TAS2R38 gene: PAV/PAV, PAV/AVI and AVI/AVI, at genotyping (further details are given in online supplemental file 1).

    Supplemental material

    Study visits occurred three times a year for up to 10 years (September 2011 to February 2022). The current analysis was limited to patients who had at least five study visits consecutively scheduled (in order to avoid bias from the dropout of patients with a more favourable outcome) and who were able successfully to perform nNO and lung function measurements.25–28 Informed consent was obtained from adult patients or from the parents of children, and age-appropriate assent from the children themselves. The study protocol was approved by the local hospital ethical committee and was part of the study ‘Primary Ciliary Dyskinesia (PCD): diagnosis, clinical phenotypes and prevalence in an Italian pediatric population’ (2644P).

    Study procedures

    At each follow-up visit, standardised protocols were employed for the assessment of clinical features including medical history, physical examination and measurement of nNO (CLD 88 Exhalyzer; EcoMedics, Duernten, Switzerland), performed according to standard methodologies.25 26 We also performed plethysmography followed by spirometry (Master Screen Body equipment; Jaeger), in accord with European Respiratory Society/American Thoracic Society criteria.27 28 Further details are given in the online supplemental file 1. We collected expectorated sputum or pharyngeal aspirates as appropriate for microbiological analysis. Each evaluation was performed when subjects had been free from signs and symptoms of acute respiratory infection for ≥4 weeks. P.a. infection was defined as chronic when patients had at least 2 (more than 50%) positive sputum or pharyngeal aspirates cultures during the previous 12 months.21 29 Forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC ratio and forced expiratory flow between 25% and 75% of vital capacity (FEF25–75) z-scores, as well as functional residual capacity (FRC), residual volume (RV), total lung capacity (TLC) and RV/TLC z-scores were calculated from the Global Lung Initiative reference values.30 31

    Statistical analysis

    The sample size was opportunistic, in view of the lack of data to inform a power calculation. Descriptive statistics are presented for the entire cohort, and for the three most frequent genetic groups (CCDC39/CCDC40, DNAH5 and DNAH11). Fisher’s exact test and t-test were used to compare baseline cross-sectional data (table 1). We used mixed effects models for all repeated measurements from each participant, including random intercept and random slope, to estimate the age-related changes in nNO. TAS2R38 polymorphisms in the overall study population, and within the three most frequent genetic groups were included as fixed effects3 5 in our models to examine their specific effects on the outcome variable (tables 2 and 3). We also evaluated the association between TAS2R38 haplotypes and longitudinal lung function parameters, and estimated the age-related changes in lung function parameters only in those genetic group(s) who were found to have significant differences between mean trends of nNO in relation to the polymorphism (table 4). We also used mixed-effects logistic regression models to estimate the age-related occurrence of P.a. infections (table 2). Finally, we evaluated any associations between number of children infected, age of first infection, prevalence of chronic pulmonary infection with P.a. and polymorphisms or at least one PAV allele by using Fisher’s exact test.

    View this table:
    Table 1

    Overall demographic and clinical features by genotypes and TAS2R38 polymorphisms

    View this table:
    Table 2

    Estimated mean annual change in nNO production (nL/min) and in number of P.a. infections by TAS2R38 polymorphism and genotype

    View this table:
    Table 3

    Mean trend of nNO over time by TAS2R38 polymorphism and genotype

    View this table:
    Table 4

    Estimated mean annual change in spirometric and plethysmographic parameters by TAS2R38 polymorphism in DNAH11 group

    A p value<0.05 was considered statistically significant. All statistical calculations were performed using R V.3.5.3.

    Results

    119 patients with PCD aged >5 years met the inclusion criteria and were enrolled in the study (55 children and 64 adults). There were 1116 study visits, mean 9 per patient (range 5–20); 27 children (5–18 years old) became adults (>18 years old) during the follow-up period. Mean (range) follow-up was 5.7 (2–10) years.

    Genetic mutations (two pathogenic variants in a known PCD-associated gene, or one pathogenic variant in the X chromosome in men, for X-linked cases) are reported in online supplemental table 1. Genetic findings were confirmed by ultrastructural investigation, ciliary motion analysis and ciliogenesis results (data not shown).

    Supplemental material

    Baseline patient characteristics

    Overall baseline demographic and clinical characteristics including nNO values and P.a. infection status, PCD genotype and TAS2R38 polymorphisms are shown in table 1. In particular, the prevalence of the PAV/PAV haplotype in the whole cohort of patients with PCD was 26%, while that of PAV/AVI and AVI/AVI was 53% and 21%, respectively, as expected from previous studies.21 32 33

    We assessed any nasal polyposis and sinus surgery occurring before the start of the study as possibly influencing nNO measurements, but no patient subsequently suffered these complications or required sinus surgery. There was no statistically significant difference between the variables measured in the different groups, except that nNO values at enrolment were lower in the combined CCDC39 and CCDC40 group (together) compared with the DNAH11 group (p=0.025).

    Longitudinal changes in nasal nitric oxide in relation to TAS2R38 polymorphisms

    The estimated mean annual change and mean trend in nNO production over time for each TAS2R38 polymorphism for the cohort as a whole and each genotype are shown in tables 2 and 3, respectively. Table 2 shows no statistically significant mean annual change of nNO production in any TAS2R38 polymorphism group. Moreover, there were no significant differences in mean trend of nNO between the three TAS2R38 polymorphism groups for the whole cohort and for each genotype (table 3). The one exception was patients with PAV/PAV haplotype and pathogenic mutations in the DNAH11 gene (figure 1). In this group, the mean trend of nNO production was significantly higher than in patients with AVI/AVI haplotype (p=0.033). However, mean values of nNO were usually higher (although not significantly) in patients with the PAV allele (higher in PAV/PAV than in PAV/AVI, itself higher than in AVI/AVI), regardless of genotype (table 3).

    Figure 1
    Figure 1

    Forest plots of association of nNO levels and TAS2R38 polymorphisms in patients with DNAH11 mutations: PAV/AVI (reference group, n=11), PAV/PAV (n=9), AVI/AVI (n=8). Linear mixed effects models including data from all study visits were used to estimate the overall association between nNO levels and haplotypes. Thus, the mean trend of nNO production was significantly higher in patients with PAV/PAV haplotype than in those with AVI/AVI haplotype (p=0.033). nNO, nasal nitric oxide.

    Longitudinal changes of lung function parameters in relation to TAS2R38 polymorphisms as a clinical outcome measure of patients with PCD with DNAH11 mutations

    We assessed the mean annual change and the mean trends of spirometric and plethysmographic parameters over the total time of the study period in the DNAH11 group, comparing the patients with the three different TAS2R38 haplotypes.

    The mean annual changes in most spirometric parameters (table 4) were not significant, but were always better in the PAV/PAV haplotype; the FEF25–75 z-score alone was significantly better (p=0.042). By contrast, the mean annual increase in RV and RV/TLC z-score was significant in all haplotypes, but greater in AVI/AVI than in AVI/PAV and in the latter group higher than in PAV/PAV (table 4). The rise in RV and RV/TLC implies progressively worsening air trapping and hyperinflation. Furthermore, most of the mean trends of spirometric and plethysmographic parameters over the total time of the study period were significantly better in patients with PAV/PAV haplotype than in the others, as shown in figure 2A,B (additional results in online supplemental table 2, online supplemental figure 1).

    Supplemental material

    Supplemental material

    Figure 2
    Figure 2

    (A) Forest plots of association of spirometric parameters (FEV1 and FEV1/FVC z score) and TAS2R38 polymorphisms in patients with DNAH11 mutations: PAV/AVI (reference group, n=11), PAV/PAV (n=9), AVI/AVI (n=8). Mean trends of FEV1 z score over the total time of the study period were significantly better in patients with PAV/PAV haplotype than in those with PAV/AVI (p=0.005) and AVI/AVI haplotype (p=0.001). Likewise, mean trends of FEV1/FVC z score were significantly better in patients with PAV/PAV haplotype than in those with PAV/AVI (p<0.0001) and AVI/AVI (p<0.0001). (B) Forest plots of association of plethysmographic parameters (RV and RV/TLC z score) and TAS2R38 polymorphisms in patients with DNAH11 mutations: PAV/AVI (reference group, n=11), PAV/PAV (n=9), AVI/AVI (n=8). Mean trends of RV z score over the total time of the study period were significantly better in patients with PAV/PAV haplotype than in those with AVI/AVI (p<0.0001) and in patients with PAV/AVI than in those with AVI/AVI (p<0.0001). Likewise, mean trends of RV/TLC z score were significantly better in patients with PAV/PAV haplotype than in those with PAV/AVI (p=0.007) and AVI/AVI (p<0.0001). Moreover, mean trends of RV/TLC z score were significantly better in patients with PAV/AVI haplotype than in those with AVI/AVI (p=0.009). Linear mixed effects models including data from all study visits were used to estimate the overall association between lung function and haplotypes. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; RV, residual volume; TLC, total lung capacity.

    TAS2R38 polymorphisms and P.a. infections over time

    Respiratory cultures were from expectorated sputum (98.8%) and pharyngeal aspirates (1.2%) at 993 of the 1116 (89%) study visits in the 119 enrolled patients. We acknowledge that sputum may be more sensitive, but we used deep pharyngeal aspiration to try to minimise any differences. Table 2 shows the estimated mean annual change in the number of P.a. infections for each TAS2R38 polymorphism for the cohort as a whole and each of the three PCD genotypes studied individually (above). Those patients from the whole cohort with PAV/AVI and PAV/PAV haplotypes had a significant mean annual increase in these infections (p<0.001 and p=0.01, respectively). This was also true for the subgroup with DNAH5 genotype (PAV/AVI p<0.001 and PAV/PAV p=0.03, respectively). Moreover, both in the whole cohort and in each genotype, the TAS2R38 polymorphism type showed no significant association with the number of patients having at least one P.a. infection (table 1). However, in the group of patients with pathogenic mutations in the DNAH11 gene, the PAV allele (PAV/AVI and PAV/PAV haplotypes together) was associated with a significantly reduced prevalence of chronic P.a. infection (p=0.029). Finally, although the age of the first P.a. infection in the whole cohort of children was not significantly different between the TAS2R38 polymorphism groups (table 1), in children the PAV allele was again associated with a significantly reduced number of P.a. isolates (p=0.046).

    Discussion

    In our longitudinal, single-centre study of a large cohort of PCD adult and paediatric patients, we describe for the first time the long-term evolution of nNO, spirometry and plethysmography in relation to TAS2R38 polymorphisms (PAV/PAV, PAV/AVI, AVI/AVI) and to lower airways P.a. infection. We combined all patients with PCD irrespective of genotype, and also separately studied the three the most common genetic groups (CCDC39/CCDC40, DNAH5 and DNAH11). We demonstrated that only patients with PCD with DNAH11 mutations had a mean trend of nNO production significantly higher in those with the PAV/PAV haplotype than AVI/AVI haplotype and this was associated with a better mean trend in lung function in the PAV/PAV group. Furthermore, we also confirmed that in the same patients the PAV allele was associated with a significantly reduced prevalence of chronic P.a. infection, as some,23 but not all22 have described in cystic fibrosis. The prevalence of TAS2R38 polymorphisms in our cohort of patients with PCD was similar to those in a Caucasian population32 and in a small series of patients with PCD studied at another Italian centre.21 33 Likewise, the distribution of polymorphisms was also as expected in our three most frequent PCD genotype groups.

    These results need to be interpreted cautiously, given that the numbers of patients with each TAS2R38 polymorphism are small and a type 1 error cannot be excluded. Confirmation in a larger study is needed. Nevertheless, it is possible to speculate that in a relatively mild phenotype PCD group like DNAH11 patients, the effect of modifier genes may be important, whereas in patients with more severe PCD, for example, CCDC39/CCDC40, other factors promoting disease severity may drown out any modifier gene signal. However, the fact that no such associations were seen in another group of comparable size, but with less respiratory impairment,3–6 DNAH5, although with only four AVI/AVI patients, means that this interpretation must be considered highly speculative.

    Several previous studies have described the relationship between the altered function of the receptor for bitter taste (due to the lack of the PAV allele) and upper respiratory infections, chronic rhinosinusitis, nasal polyposis requiring surgery or chronic pulmonary infection by P.a. also in childhood, particularly in cystic fibrosis patients.14 23 33–37 Thus, it has been suggested that TAS2R38 may be a novel disease modifier gene.23 Although TAS2R38 polymorphisms in CCDC39/CCDC40 patients did not relate to mean trends in nNO production over time, in patients with mutations in the DNAH11, a group in which a less severe clinical course would be anticipated, the PAV/PAV haplotype was associated with a mean trend in nNO production over time which was significantly higher than in patients with AVI/AVI haplotype. This was associated with a better mean trend in lung function over time in the PAV/PAV compared with the AVI/AVI groups. In particular, we believe that the differences between the two groups in plethysmographic parameters are likely clinically significant, as they better predict high-resolution CT (HRCT) abnormalities than spirometry38 and suggest a milder clinical course of PAV/PAV than AVI/AVI haplotype. HRCT were only performed to guide therapy in patients who had an adverse course, so we cannot use this investigation to confirm our findings. However, confirmatory evidence of the lung function findings is that, although both in the whole cohort and in each genotype group the TAS2R38 polymorphism type showed no significant association with the number of patients having at least one P.a. isolate, in patients with biallelic DNAH11 mutations the PAV allele was associated with a significantly lower prevalence of chronic P.a. infection.

    Recently, we have demonstrated that patients with PCD with pathogenic mutations in the CCDC39/CCDC40 genes had the steepest decline over time in nNO and a higher prevalence of P.a. infections compared with the other most frequent genotypes.7 In the present study, at enrolment, nNO values in the CCDC39/CCDC40 group were significantly lower than in patients with mutations in DNAH11. Hence the lower levels of nNO in patients with mutations in CCDC39/CCDC40 cannot be linked to TAS2R38 haplotype.

    Particularly, in DNAH11 patients with less impaired lung function and generally better prognosis,2–7 we speculate that the genotypic differences in the bitter taste receptor may be important in innate antimicrobial effects influencing the immune response to P.a. infections. This might account for the phenotypic variability of the disease in patients with DNAH11 mutations because there is a lower risk of chronic infections if the PAV allele is present. In these patients, we speculate that the presence of at least one PAV allele in the bitter taste receptor T2R38 in airway cilia might exhibit enhanced activation by quorum-sensing molecules secreted by P.a. leading to calcium-dependent nitric oxide production.14–16 This would likely not lead to a reduced prevalence of infection per se, but rather reduced progression to chronicity. In support of this speculation, no patients with PCD in a small series who were homozygous for PAV/PAV (unreported PCD genotype) were found to be chronically infected with P.a.21 Likewise, the significant mean annual increase of P.a. infection in our patients with mutations in DNAH5 gene, where the PAV allele was not associated with similar trends in nNO production, is supportive.

    Moreover, although the age of first P.a. infection in children was not significantly different in TAS2R38 polymorphisms regardless of PCD causing genes, in our whole cohort of children with PCD the PAV allele was also associated with a significantly lower number of P.a. infections, further suggesting its potential protective role in this age group.

    The strengths of our study include its prospective, longitudinal, single-centre design and the inclusion of a large cohort of all age patients with PCD diagnosed using standardised criteria.23 A single-centre study has reduced variability in study procedures, in treatments (such as twice daily nasal rinses, respiratory physiotherapy and chronic administration of azithromycin in all patients with PCD and inhaled anti-P.a. antibiotics in all P.a.-chronically infected patients with PCD as reported in online supplemental table 3), and in nNO measurements, since the same standard operating procedures are always used. Moreover, as in this study, it is important to consider subgroups in modifier gene studies, otherwise significant effects may be missed. However, this does of course mean there may be only small numbers in some or all subgroups.

    Supplemental material

    Our study has some weaknesses. The first is that, due to the large numbers of genes involved in ciliary assembly and function, the sample size for each genetic subgroup is small and many genotypes could not be studied due to small numbers. Thus, we could obtain information on the time course of nNO production and of P.a. infections in relation to TAS2R38 polymorphisms only in the three most frequent genotypes. Indeed, even in the three major gene groups we studied separately, the numbers with each TAS2R38 alleles are very small, thus our study must be considered as hypothesis generating. Second, nNO levels in patients with PCD are not only an expression of the activation of T2R38 receptors by P.a. quorum-sensing molecules. Indeed, NO is a product of L-arginine metabolism by one of three isoforms of nitric oxide synthase (NOS) and the cause for the low levels of nNO in PCD is still unknown, nor is it clear which NOS isoenzyme synthesises most nNO in vivo. NOS2 and NOS3 were found in nasal epithelial cells,39 but NOS2 gene expression was lower in patients with PCD with unreported genotype.40 Also, since variants in modifier genes as the T allele in NOS3 may play an important role in a higher nNO production, which could be protective against P.a. infections in PCD,40 a further limitation is the lack of information relating nNO of our patients to polymorphisms in the NOS3. Thus, it is possible that the variability in biological activity of NOS isoenzymes could partially explain the different rates of nNO production over time, but we did not evaluate it in the present study. Likewise, we did not study the recently reported link between high nNO values and potentially hypomorphic genetic mutations (missense, single amino acid deletion or moderate splicing mutations) with possible residual functional protein production.41 Finally, the magnitude of NO change is small and it is surprising that this would significantly influence lung function. Moreover, we were not able to triangulate our physiological data with lung structure on HRCT because regular HRCT scanning in all patients with PCD is not our unit protocol.

    Conclusion

    We have shown that in patients with DNAH11 mutations, the PAV/PAV haplotype was associated with a mean trend in nNO production significantly higher than in the AVI/AVI haplotype which was associated with a better mean trend in lung function over time. Moreover, the PAV allele was associated with a significantly lower prevalence of chronic P.a. infection. Although the numbers are small, the fact that the conclusion is supported by three different tests (nNO, P.a. infection and lung function) is striking evidence of plausibility. This was not the case in patients with CCDC39/CCDC40 and DNAH5 mutations. Therefore, the differences in bitter taste receptors are likely only important in those patients with less impaired lung function and generally better prognosis. These results require confirmation in more extensive multicentre studies on TAS2R38 polymorphisms. Such studies might help to improve management strategies by more aggressive or personalised treatment to prevent chronic P.a. infections, mostly in patients with PCD with an elevated risk.

    Data availability statement

    Data are available upon reasonable request. De-identified participant data are available from the corresponding author upon reasonable request, subject to the terms of Ethics Committee approval.

    Ethics statements

    Patient consent for publication

    Ethics approval

    This study involves human participants and was approved by Pisa Hospital Ethical Committee, reference number 2644P. Participants gave informed consent to participate in the study before taking part.

    Acknowledgments

    The authors thank the patients with primary ciliary dyskinesia ( PCD) and/or their parents for participation in the study. We also express our gratitude to Letizia Andolfi, President, and Gaspare Di Maria, Sara Bellu, Maria Lucia Baldari and Silvana Gentile, Board Members of Italian PCD – Kartagener Syndrome Association for constant support to our research. This work was generated within the European Reference Network for Rare Respiratory Diseases (ERN-LUNG).

    References

    1. ↵
      2. Bush A ,
      3. Hogg C
      . Primary ciliary dyskinesia: recent advances in epidemiology, diagnosis, management and relationship with the expanding spectrum of ciliopathy. Expert Rev Respir Med 2012;6:663–82. doi:10.1586/ers.12.60
      OpenUrlCrossRefPubMed
    2. ↵
      2. Davis SD ,
      3. Ferkol TW ,
      4. Rosenfeld M , et al
      . Clinical features of childhood primary ciliary dyskinesia by genotype and ultrastructural phenotype. Am J Respir Crit Care Med 2015;191:316–24. doi:10.1164/rccm.201409-1672OC
      OpenUrlCrossRefPubMed
    3. ↵
      2. Davis SD ,
      3. Rosenfeld M ,
      4. Lee H-S , et al
      . Primary Ciliary Dyskinesia: Longitudinal Study of Lung Disease by Ultrastructure Defect and Genotype. Am J Respir Crit Care Med 2019;199:190–8. doi:10.1164/rccm.201803-0548OC
      OpenUrlCrossRefPubMed
    4. ↵
      2. Pifferi M ,
      3. Bush A ,
      4. Mariani F , et al
      . Lung Function Longitudinal Study by Phenotype and Genotype in Primary Ciliary Dyskinesia. Chest 2020;158:117–20. doi:10.1016/j.chest.2020.02.001
      OpenUrlCrossRefPubMed
    5. ↵
      2. Pifferi M ,
      3. Bush A ,
      4. Mulé G , et al
      . Longitudinal Lung Volume Changes by Ultrastructure and Genotype in Primary Ciliary Dyskinesia. Ann Am Thorac Soc 2021;18:963–70. doi:10.1513/AnnalsATS.202007-816OC
      OpenUrl
    6. ↵
      2. Shoemark A ,
      3. Rubbo B ,
      4. Legendre M , et al
      . Topological data analysis reveals genotype-phenotype relationships in primary ciliary dyskinesia. Eur Respir J 2021;58:2002359. doi:10.1183/13993003.02359-2020
    7. ↵
      2. Pifferi M ,
      3. Boner AL ,
      4. Gracci S , et al
      . Longitudinal Nitric Oxide Levels and Infections by Ultrastructure and Genotype in Primary Ciliary Dyskinesia. Chest 2022;162:1265–76. doi:10.1016/j.chest.2022.06.019
      OpenUrl
    8. ↵
      2. Shapiro AJ ,
      3. Josephson M ,
      4. Rosenfeld M , et al
      . Accuracy of Nasal Nitric Oxide Measurement as a Diagnostic Test for Primary Ciliary Dyskinesia. A Systematic Review and Meta-analysis. Ann Am Thorac Soc 2017;14:1184–96. doi:10.1513/AnnalsATS.201701-062SR
      OpenUrl
    9. ↵
      2. Shapiro AJ ,
      3. Davis SD ,
      4. Leigh MW , et al
      . Limitations of Nasal Nitric Oxide Testing in Primary Ciliary Dyskinesia. Am J Respir Crit Care Med 2020;202:476–7. doi:10.1164/rccm.202003-0835LE
      OpenUrl
    10. ↵
      2. O’Connor MG ,
      3. Horani A ,
      4. Shapiro AJ
      . Progress in Diagnosing Primary Ciliary Dyskinesia: The North American Perspective. Diagnostics (Basel) 2021;11:1278. doi:10.3390/diagnostics11071278
    11. ↵
      2. Knowles MR ,
      3. Ostrowski LE ,
      4. Leigh MW , et al
      . Mutations in RSPH1 Cause Primary Ciliary Dyskinesia with a Unique Clinical and Ciliary Phenotype. Am J Respir Crit Care Med 2014;189:707–17. doi:10.1164/rccm.201311-2047OC
      OpenUrlCrossRefPubMedWeb of Science
    12. ↵
      2. Shoemark A ,
      3. Moya E ,
      4. Hirst RA , et al
      . High prevalence of CCDC103 p.His154Pro mutation causing primary ciliary dyskinesia disrupts protein oligomerisation and is associated with normal diagnostic investigations. Thorax 2018;73:157–66. doi:10.1136/thoraxjnl-2017-209999
      OpenUrlAbstract/FREE Full Text
    13. ↵
      2. Raidt J ,
      3. Krenz H ,
      4. Tebbe J , et al
      . Limitations of Nasal Nitric Oxide Measurement for Diagnosis of Primary Ciliary Dyskinesia with Normal Ultrastructure. Ann Am Thorac Soc 2022;19:1275–84. doi:10.1513/AnnalsATS.202106-728OC
      OpenUrl
    14. ↵
      2. Lee RJ ,
      3. Xiong G ,
      4. Kofonow JM , et al
      . T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection. J Clin Invest 2012;122:4145–59. doi:10.1172/JCI64240
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      2. Lee RJ ,
      3. Cohen NA
      . Taste receptors in innate immunity. Cell Mol Life Sci 2015;72:217–36. doi:10.1007/s00018-014-1736-7
      OpenUrlCrossRefPubMed
    16. ↵
      2. Carey RM ,
      3. Lee RJ
      . Taste Receptors in Upper Airway Innate Immunity. Nutrients 2019;11:2017. doi:10.3390/nu11092017
    17. ↵
      2. Bufe B ,
      3. Breslin PAS ,
      4. Kuhn C , et al
      . The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception. Curr Biol 2005;15:322–7. doi:10.1016/j.cub.2005.01.047
      OpenUrlCrossRefPubMedWeb of Science
    18. ↵
      2. Floriano WB ,
      3. Hall S ,
      4. Vaidehi N , et al
      . Modeling the human PTC bitter-taste receptor interactions with bitter tastants. J Mol Model 2006;12:931–41. doi:10.1007/s00894-006-0102-6
      OpenUrl
    19. ↵
      2. Freund JR ,
      3. Lee RJ
      . Taste receptors in the upper airway. World J Otorhinolaryngol Head Neck Surg 2018;4:67–76. doi:10.1016/j.wjorl.2018.02.004
      OpenUrl
    20. ↵
      2. Boxer EE ,
      3. Garneau NL
      . Rare haplotypes of the gene TAS2R38 confer bitter taste sensitivity in humans. Springerplus 2015;4:505. doi:10.1186/s40064-015-1277-z
    21. ↵
      2. Piatti G ,
      3. Ambrosetti U ,
      4. Robino A , et al
      . Primary Ciliary Dyskinesia: The Impact of Taste Receptor (TAS2R38) Gene Polymorphisms on Disease Outcome and Severity. Int Arch Allergy Immunol 2020;181:727–31. doi:10.1159/000508938
      OpenUrl
    22. ↵
      2. Turnbull AR ,
      3. Murphy R ,
      4. Behrends V , et al
      . Impact of T2R38 Receptor Polymorphisms on Pseudomonas aeruginosa Infection in Cystic Fibrosis. Am J Respir Crit Care Med 2018;197:1635–8. doi:10.1164/rccm.201711-2365LE
      OpenUrlCrossRef
    23. ↵
      2. Castaldo A ,
      3. Cernera G ,
      4. Iacotucci P , et al
      . TAS2R38 is a novel modifier gene in patients with cystic fibrosis. Sci Rep 2020;10:5806. doi:10.1038/s41598-020-62747-9
    24. ↵
      2. Lucas JS ,
      3. Barbato A ,
      4. Collins SA , et al
      . European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia. Eur Respir J 2017;49:1601090. doi:10.1183/13993003.01090-2016
    25. ↵
      ATS/ERS Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide, 2005. Am J Respir Crit Care Med 2005;171:912–30. doi:10.1164/rccm.200406-710ST
      OpenUrlCrossRefPubMedWeb of Science
    26. ↵
      2. Shapiro AJ ,
      3. Dell SD ,
      4. Gaston B , et al
      . Nasal Nitric Oxide Measurement in Primary Ciliary Dyskinesia. A Technical Paper on Standardized Testing Protocols. Ann Am Thorac Soc 2020;17:e1–12. doi:10.1513/AnnalsATS.201904-347OT
      OpenUrl
    27. ↵
      2. Wanger J ,
      3. Clausen JL ,
      4. Coates A , et al
      . Standardisation of the measurement of lung volumes. Eur Respir J 2005;26:511–22. doi:10.1183/09031936.05.00035005
      OpenUrlAbstract/FREE Full Text
    28. ↵
      2. Miller MR ,
      3. Hankinson J ,
      4. Brusasco V
      . Standardisation of spirometry. Eur Respir J 2005;26:319–38. doi:10.1183/09031936.05.00034805
      OpenUrlAbstract/FREE Full Text
    29. ↵
      2. Lee TWR ,
      3. Brownlee KG ,
      4. Conway SP , et al
      . Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J Cyst Fibros 2003;2:29–34. doi:10.1016/S1569-1993(02)00141-8
      OpenUrlCrossRefPubMed
    30. ↵
      2. Quanjer PH ,
      3. Stanojevic S ,
      4. Cole TJ , et al
      . Multi-ethnic reference values for spirometry for the 3-95-yr age range: the global lung function 2012 equations. Eur Respir J 2012;40:1324–43. doi:10.1183/09031936.00080312
      OpenUrlAbstract/FREE Full Text
    31. ↵
      2. Hall GL ,
      3. Filipow N ,
      4. Ruppel G , et al
      . Official ERS technical standard: Global Lung Function Initiative reference values for static lung volumes in individuals of European ancestry. Eur Respir J 2021;57:2000289. doi:10.1183/13993003.00289-2020
    32. ↵
      2. Kim UK ,
      3. Drayna D
      . Genetics of individual differences in bitter taste perception: lessons from the PTC gene. Clin Genet 2005;67:275–80. doi:10.1111/j.1399-0004.2004.00361.x
      OpenUrlCrossRefPubMedWeb of Science
    33. ↵
      2. Piatti G ,
      3. Ambrosetti U ,
      4. Aldè M , et al
      . Chronic Rhinosinusitis: T2r38 Genotyping and Nasal Cytology in Primary Ciliary Dyskinesia. Laryngoscope 2023;133:248–54. doi:10.1002/lary.30112
      OpenUrl
    34. ↵
      2. Lee RJ ,
      3. Cohen NA
      . Role of the bitter taste receptor T2R38 in upper respiratory infection and chronic rhinosinusitis. Curr Opin Allergy Clin Immunol 2015;15:14–20. doi:10.1097/ACI.0000000000000120
      OpenUrlCrossRefPubMed
    35. ↵
      2. Adappa ND ,
      3. Farquhar D ,
      4. Palmer JN , et al
      . TAS2R38 genotype predicts surgical outcome in nonpolypoid chronic rhinosinusitis. Int Forum Allergy Rhinol 2016;6:25–33. doi:10.1002/alr.21666
      OpenUrlCrossRefPubMed
    36. ↵
      2. Cohen NA
      . The genetics of the bitter taste receptor T2R38 in upper airway innate immunity and implications for chronic rhinosinusitis. Laryngoscope 2017;127:44–51. doi:10.1002/lary.26198
      OpenUrl
    37. ↵
      2. Yilmaz G ,
      3. Eyigor H ,
      4. Gur OE , et al
      . The role of TAS2R38 genotype in surgical outcomes and culturable bacteria in chronic rhinosinusitis with or without nasal polyps. Rhinology 2023;61:54–60. doi:10.4193/Rhin22.118
      OpenUrl
    38. ↵
      2. Pifferi M ,
      3. Bush A ,
      4. Pioggia G , et al
      . Evaluation of pulmonary disease using static lung volumes in primary ciliary dyskinesia. Thorax 2012;67:993–9. doi:10.1136/thoraxjnl-2011-200137
      OpenUrlAbstract/FREE Full Text
    39. ↵
      2. Petruson K ,
      3. Stalfors J ,
      4. Jacobsson KE , et al
      . Nitric oxide production in the sphenoidal sinus by the inducible and constitutive isozymes of nitric oxide synthase. Rhinology 2005;43:18–23.
      OpenUrlPubMedWeb of Science
    40. ↵
      2. Pifferi M ,
      3. Bush A ,
      4. Maggi F , et al
      . Nasal nitric oxide and nitric oxide synthase expression in primary ciliary dyskinesia. Eur Respir J 2011;37:572–7. doi:10.1183/09031936.00044310
      OpenUrlAbstract/FREE Full Text
    41. ↵
      2. Legendre M ,
      3. Thouvenin G ,
      4. Taytard J , et al
      . High Nasal Nitric Oxide, Cilia Analyses, and Genotypes in a Retrospective Cohort of Children with Primary Ciliary Dyskinesia. Ann Am Thorac Soc 2022;19:1704–12. doi:10.1513/AnnalsATS.202110-1175OC
      OpenUrl

    Supplementary materials

    Footnotes

    • Contributors MPif, ABo, DM and ABu designed the study, analysed all data, wrote the manuscript. MPif, DM, AM, AMC, RG, GB, MPia and VB collected the experimental data, and contributed to data evaluation. GD and CD participated in processing data and performed the statistical analysis. MAC, AV and DP contributed to interpretation of data, and helped to draft the manuscript for important intellectual content. All authors revised the report and approved the final version before submission. MPif is the guarantor of the article and all authors take responsibility for the content of the manuscript, the integrity of the data and the accuracy of the data analysis.

    • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

    Linked Articles