Standardizing nasal nitric oxide measurement as a test for primary ciliary dyskinesia

Abstract

Rationale: Several studies suggest that nasal nitric oxide (nNO) measurement could be a test for primary ciliary dyskinesia (PCD), but the procedure and interpretation have not been standardized.

Objectives: To use a standard protocol for measuring nNO to establish a disease-specific cutoff value at one site, and then validate at six other sites.

Methods: At the lead site, nNO was prospectively measured in individuals later confirmed to have PCD by ciliary ultrastructural defects (n = 143) or DNAH11 mutations (n = 6); and in 78 healthy and 146 disease control subjects, including individuals with asthma (n = 37), cystic fibrosis (n = 77), and chronic obstructive pulmonary disease (n = 32). A disease-specific cutoff value was determined, using generalized estimating equations (GEEs). Six other sites prospectively measured nNO in 155 consecutive individuals enrolled for evaluation for possible PCD.

Measurements and main results: At the lead site, nNO values in PCD (mean ± standard deviation, 20.7 ± 24.1 nl/min; range, 1.5-207.3 nl/min) only rarely overlapped with the nNO values of healthy control subjects (304.6 ± 118.8; 125.5-867.0 nl/min), asthma (267.8 ± 103.2; 125.0-589.7 nl/min), or chronic obstructive pulmonary disease (223.7 ± 87.1; 109.7-449.1 nl/min); however, there was overlap with cystic fibrosis (134.0 ± 73.5; 15.6-386.1 nl/min). The disease-specific nNO cutoff value was defined at 77 nl/minute (sensitivity, 0.98; specificity, >0.999). At six other sites, this cutoff identified 70 of the 71 (98.6%) participants with confirmed PCD.

Conclusions: Using a standardized protocol in multicenter studies, nNO measurement accurately identifies individuals with PCD, and supports its usefulness as a test to support the clinical diagnosis of PCD.

Figure 1.

Scatter plot of nasal nitric oxide (nNO) values (natural log scale; nl/min) versus age for individuals with primary ciliary dyskinesia (PCD) and healthy control subjects, using model-predicted values and a 95% confidence band. For nNO measurements in subjects with PCD and ciliary ultrastructure defects evaluated at the University of North Carolina site (open triangles, single measurements; solid triangles, repeated measurements), no trends with respect to age were observed; consequently, an intercept-only model adequately captured the variation in nNO levels. For healthy control subjects (open circles), nNO increased with age in a nonlinear fashion; consequently, the model that appeared to provide the best fit included separate linear slopes with respect to log_e age for subjects between 5 and 11 years and subjects older than 11 years of age.

Figure 2.

Scatter plot of nasal nitric oxide (nNO) values (linear scale; nl/min) versus age for individuals with primary ciliary dyskinesia (PCD) and healthy control subjects with nNO cutoff (same data as in Figure 1, shown on a linear scale). All nNO values from healthy control subjects (open circles) were well above the cutoff (77 nl/min) and most of the nNO measurements in subjects with PCD and ciliary ultrastructure defects (open triangles, single measurements; solid triangles, repeated measurements) were below the cutoff. The three solid triangles above the cutoff are repeated measurements in the same individual with PCD.

Figure 3.

Scatter plot of nasal nitric oxide (nNO) values (linear scale; nl/min) versus age for University of North Carolina disease control subjects and a subset of subjects with primary ciliary dyskinesia (PCD) with normal electron micrographs but biallelic mutations in DNAH11. Individual nNO measurements are shown for disease control subjects: asthma (open circles), cystic fibrosis (open squares), and chronic obstructive pulmonary disease (solid triangles). In addition, 10 measurements are shown for a special subset of PCD (solid circles) with PCD confirmed by the presence of biallelic mutations in DNAH11 despite normal ultrastructure.

Figure 4.

Validation data from six other sites: primary ciliary dyskinesia (PCD) confirmed with ultrastructural defect or biallelic mutations in gene-associated electron microscopy (EM) defect. Single nasal nitric oxide (nNO) measurements are shown for the six other Genetic Disorders of Mucociliary Clearance Consortium (GDMCC) sites: National Institute of Allergy and Infectious Diseases (Bethesda, MD; 43 participants); Washington University (Saint Louis, MO; 39 participants); Hospital for Sick Children (Toronto, ON, Canada; 26 participants); University of Colorado (Denver, CO; 23 participants); University of Washington (Seattle, WA; 18 participants); and Stanford University (Palo Alto, CA; 6 participants). The open circles represent individuals with PCD confirmed by identification of ultrastructural defect. The solid circles represent individuals with PCD confirmed by genetic testing alone, demonstrating biallelic mutations in a PCD gene.

Figure 5.

Validation data from six other sites: Participants undergoing primary ciliary dyskinesia (PCD) evaluation but no confirmatory ultrastructural defect or genetic mutations. The triangles represent individuals with a laterality defect (situs inversus totalis or heterotaxy) and the circles represent individuals with PCD with no laterality defect. Participants represented by the open symbols have low symptom scores (0–2) and participants represented by the solid symbols have high symptom scores (3 or 4).