High prevalence of CCDC103 p.His154Pro mutation causing primary ciliary dyskinesia disrupts protein oligomerisation and is associated with normal diagnostic investigations

Abstract

Rationale: Primary ciliary dyskinesia is a genetically heterogeneous inherited condition characterised by progressive lung disease arising from abnormal cilia function. Approximately half of patients have situs inversus. The estimated prevalence of primary ciliary dyskinesia in the UK South Asian population is 1:2265. Early, accurate diagnosis is key to implementing appropriate management but clinical diagnostic tests can be equivocal.

Objectives: To determine the importance of genetic screening for primary ciliary dyskinesia in a UK South Asian population with a typical clinical phenotype, where standard testing is inconclusive.

Methods: Next-generation sequencing was used to screen 86 South Asian patients who had a clinical history consistent with primary ciliary dyskinesia. The effect of a CCDC103 p.His154Pro missense variant compared with other dynein arm-associated gene mutations on diagnostic/phenotypic variability was tested. CCDC103 p.His154Pro variant pathogenicity was assessed by oligomerisation assay.

Results: Sixteen of 86 (19%) patients carried a homozygous CCDC103 p.His154Pro mutation which was found to disrupt protein oligomerisation. Variable diagnostic test results were obtained including normal nasal nitric oxide levels, normal ciliary beat pattern and frequency and a spectrum of partial and normal dynein arm retention. Fifteen (94%) patients or their sibling(s) had situs inversus suggesting CCDC103 p.His154Pro patients without situs inversus are missed.

Conclusions: The CCDC103 p.His154Pro mutation is more prevalent than previously thought in the South Asian community and causes primary ciliary dyskinesia that can be difficult to diagnose using pathology-based clinical tests. Genetic testing is critical when there is a strong clinical phenotype with inconclusive standard diagnostic tests.

Keywords: CCDC103; cilia; diagnosis; genetic testing.; mutation; primary ciliary dyskinesia; respiratory tract.

Figure 1. Comparison of predicted FEV1 in CCDC103 p.His154Pro cases versus a comparator group

The CCDC103 p.His154Pro cases are described in Table 1 and the comparator group are South Asian origin CCDC103 p.His154Pro-negative cases with a confirmed absent dynein arms defect.

Figure 2. Transmission electron microscopy of CCDC103 p.His154Pro patients

Representative cilia cross sections show (A) absent outer and partially absent inner dynein arms, (B) presence of outer dynein arms only and presence of both inner and outer dynein arms within the same micrograph. (C) Inset shows presence of outer but not inner dynein arms. Black arrows indicate example outer dynein arms. White arrows indicate inner dynein arms. Scale bar, 100 nm.

Figure 3. Quantitative transmission electron microscopy survey of inner and outer dynein arm loss in CCDC103 p.His154Pro patients versus a comparator group

By surveying >100 cross sections in each patient sample we performed quantitative electron microscopy to determine the percentage of arm defects in cilia from individuals homozygous for the hypomorphic p.His154Pro CCDC103 mutation. Red diamonds and triangles indicate results from 16 CCDC103 p.His154Pro homozygote PCD patients, where triangles indicates the result of 4 repeat nasal brushings performed on patients marked by a diamond. The comparator group of 16 individuals with PCD, indicated by other symbols as shown, consists of 3 cases with DNAAF1 mutations (open diamonds), 2 DNAAF3 (black squares), 2 DNAH5 (dark blue diamonds), 3 LRRC6 (2 as light blue diamonds, 1 contained within the filled purple circle), 2 ZMYND10 (contained within the filled purple circle) and 4 cases in whom no mutations in known genes could be identified (grey squares). 6 CCDC103 p.His154Pro samples (27%) of p.His154Pro samples showed complete lack of both outer and inner dynein arms comparable to other gene mutations in the graph (purple circle). The grey shaded area represents normal range counts from >200 non PCD respiratory controls. Four CCDC103 p.His154Pro patients had counts within this normal range (one is a repeat sample (triangle) which showed similar data). Individuals with CCDC103 p.His154Pro mutation have a trend towards a distinctive pattern of partial loss of dynein arms that diverges from total dynein arm loss in the comparator group.

Figure 4. CCDC103 p.His154Pro oligomerisation capacity

Chromatograms of wildtype (red trace) and His154Pro (green trace) CCDC103 native proteins separated in a calibrated Superose 6 10/300 gel filtration column. The data is plotted as absorbance at 280 nm (in mAU) against elution volume (ml). Both proteins show strong dimer peaks at ~60 kDa. However, only the wildtype form generates a series of higher-order oligomers with an approximate mass of ~250 kDa. Aggregated material (>2 MDa) eluted in the void volume.