American Journal of Respiratory and Critical Care Medicine

Primary ciliary dyskinesia is an autosomal recessive disorder characterized by chronic upper and lower respiratory tract symptoms. We report the diagnosis of primary ciliary dyskinesia associated with a circular ciliary beat pattern in three siblings. This beat pattern is consistent with a ciliary transposition defect, where a peripheral microtubule doublet is transposed to the center of the ciliary axoneme to replace the absent central microtubule pair. However, in these siblings, ultrastructural analysis of the cilia revealed an absence of the central microtubule pair only. This variant of transposition with a circular ciliary beat pattern has not been described previously. In addition, this defect, together with the transposition defect, may help explain the mechanism of the circular beat pattern and also the absence of situs inversus in these patients.

Primary ciliary dyskinesia is an autosomal recessive disorder with an incidence of approximately 1:15,000 in the white population (1). It has been described in all major ethnic groups (1, 2). In primary ciliary dyskinesia, impaired mucociliary clearance is associated with recurrent chest infections leading to bronchiectasis and sinusitis. Symptoms frequently start in the neonatal period and include a chronic nasal discharge or nasal blockage and cough. Significant hearing problems occur in approximately 50% of children. Up to 50% of the patients may be expected to have total situs inversus (2).

Primary ciliary dyskinesia may be caused by one of a number of different ciliary ultrastructural defects, each of which results in ineffective ciliary function (2, 3). These defects include lack of inner and/or outer dynein arms, defective radial spokes, ciliary disorientation, and ciliary transposition.

In the ciliary transposition defect, in a proportion of cilia in each transmission electron microscopy cross section, the axoneme lacks the central pair of microtubules or has central microtubules extending only a short distance (3, 4). Therefore, there is a gap with no central microtubular pair. Then, in the middle and distal regions of the cilium, one peripheral microtubule doublet, with attached dynein arms, is transposed to the center of the axoneme (4). Using high-speed digital video photography, this arrangement has been shown to result in a circular movement of the cilium as opposed to the typical forward and backward planar strokes (3). The analysis of ciliary beat frequency alone may miss the diagnosis in patients with ciliary transposition, because beat frequency is often normal in this ultrastructural defect (3). The diagnosis is much less likely to be missed if beat pattern analysis and electron microscopy are undertaken in addition to beat frequency measurement. The reason for the circular beat pattern is unclear. It may be due to a lack of the central microtubular pair, or the transposed peripheral doublet that has dynein arms attached, or a combination of these abnormalities.

In this article, we report three siblings diagnosed with primary ciliary dyskinesia, associated with a circular beat pattern consistent with a ciliary transposition. In these cases, defects in the central microtubular pair occurred to a similar degree to that seen in classical transposition; however, the central microtubular pair was not replaced by a peripheral doublet. We believe this report confirms that this variant of ciliary transposition defect results in primary ciliary dyskinesia. This defect also helps to explain the circular beat pattern seen in the transposition defect.

The three siblings are of Arabic descent from the United Arab Emirates. Their parents are first cousins with four other unaffected children.

Case 1

Case 1 was a female, born at term, with an uncomplicated neonatal history, who developed a wet-sounding cough shortly after birth and had repeated hospital admissions for persistent chest infections. At the time of review, aged 5 years, she had chronic symptoms of a daily, productive cough, wheeze, and reduced exercise tolerance. She had intermittent rhinorrhoea but no apparent glue ear or ear infections. Her developmental milestones were normal. Her medications consisted of an inhaled steroid and a β-2 agonist. On examination, she was underweight (weight 2nd centile, height 10th centile). She had a wet-sounding cough and, on auscultation, had reduced air entry at the left base and basal crackles audible bilaterally. She had clear nasal discharge. On examination, both tympanic membranes were normal. There was no situs inversus.

Investigations excluded an immunoglobulin deficiency, tuberculosis, and cystic fibrosis. Cystic fibrosis was excluded by the clinical picture, a normal sweat test, and no gene mutations in a panel of 32 genes that were chosen to reflect the patient's Arabic origins. Bronchoscopy and barium swallow were normal, but a computed tomography scan demonstrated bilateral bronchiectasis and chronic left lower lobe collapse (Figure 1)

. Further investigations revealed that she had a very low nasal nitric oxide of 11.6 ppb (normal range > 200 ppb). Evaluation of her ciliary structure and function was undertaken. The nasal ciliary brush biopsy was performed and analyzed as described subsequently in Methods. Unhealthy ciliated epithelium was obtained that showed cilia beating in a circular manner at a slightly reduced frequency of 9.3 Hz (95% confidence interval: 7.9–10.6). Transmission electron microscopy revealed a central microtubule pair defect in 11.9% of cilia (95% confidence interval: 11.7–12.0).

Treatment was commenced and included physiotherapy, antibiotics, and improved nutrition. In view of her unusual biopsy findings, she had her nasal brush biopsy repeated a few months later when clinically well. Because two of her younger brothers had similar symptoms, they were also reviewed at this stage. On this occasion, none had suffered from an upper respiratory tract infection for at least 6 weeks.

Case 2

A 4-year-old boy, who was born at term, had an uncomplicated neonatal period and normal development. He had a 2-year history of a wet-sounding cough and rhinorrhoea since infancy. He was not taking any regular medications. On examination, he had diffuse bilateral rhonchi and wheeze and an offensive nasal discharge. The chest X-ray and computed tomography scan of the chest showed atelectasis in both right and left lower lobes. There was evidence of small airway disease but no bronchiectasis or dextrocardia. He was too young for assessment of nasal nitric oxide.

Case 3

The third affected sibling, a male, was reviewed when aged 8 months. He had respiratory difficulties from birth and was admitted to the special care baby unit shortly after birth. His respiratory symptoms never fully resolved, and he continued to have a wet-sounding cough and nasal symptoms. He was taking regular nebulized β-2 agonists. On examination, he was tachypnoeic with bilateral wheeze. The chest X-ray showed evidence of hyperinflation with streaky changes in the lower lobes. There was no evidence for frank bronchiectasis on the plain films. The cardiac shadow was normally sited. Again, this sibling was too young for assessment of nasal nitric oxide.

The four remaining siblings were asymptomatic and thus not assessed.

The nasal brush biopsies were performed as described previously (5). Ciliated edges greater than 50 μm in length were observed at 37°C using an oil immersion ×100 objective lens. They were recorded using a digital high-speed video camera (Kodak-Ektapro-Motion-Analyser; Eastman Kodak, San Diego, CA) at the rate of 400 frames per second. The ciliary beat frequency and ciliary beat pattern was then determined directly (5).

Brushings were fixed and prepared for transmission electron microscopy as described previously (3, 5). The microtubular and dynein arms of individual cilia from a minimum of 15 cells (370 individual cilia) were examined. The percentage of cilia with microtubular or dynein arm defects was then calculated.

The ciliated epithelium was also assessed for epithelial changes, as described previously, to determine whether there was any obvious secondary damage present in the biopsy material (5). Briefly, the number of ciliated cells, mucous cells, and dead cells was expressed as a percentage of all cells examined. Percentages were then calculated for the number of cells with loss of cilia, cellular projections, cytoplasmic blebbing, and mitochondrial damage (5).

With the exception of the first nasal ciliary biopsy obtained from the eldest sibling, healthy nasal ciliated epithelium was obtained from each case (Figure 2

, Table 1)

TABLE 1. Analysis of epithelial integrity by transmission electron microscopy



Ciliated Cells

Mucous Cells

Dead Cells

Cells with Loss of
 Cilia

Cells
 Extruding
 from Surface

Cells with
 Cytoplasmic
 Blebbing

Cells with
 Mitochondrial
 Damage

(%)
(%)
(%)
(%)
(%)
(%)
(%)
Sibling 1
First brushing69.99.00.060.43.519.310.8
Second brushing78.45.90.08.80.310.910.0
Sibling 277.04.60.00.00.010.26.9
Sibling 367.96.30.04.60.915.614.7
Normal range, %*
45.9–81.0
5.2–17.5
0.0–0.0
9.1–38.4
12.9–37.0
6.4–23.5
1.1–33.3

*Normal data for age from our laboratory, expressed as 5th to 95th percentiles (6).

Data are expressed as percentage of cells with defect present, and n = mean of 173 cells examined (range 159–204).

. The percentage of ciliated cells, dead cells, and mucous cells were all within the normal range for their age, as were the percentage of cells with loss of cilia, cellular projections, cytoplasmic blebbing, and mitochondrial damage. The ciliary beat frequency was normal in the younger siblings' biopsies and in the eldest sibling's second brush biopsy (Table 2)

TABLE 2. Analysis of ciliary function and ultrastructure in the three siblings



Ciliary Beat
 Frequency


Central
 Microtubule
 Defects

(Hz)
Ciliary Beat Pattern
(%)
Sibling 1
First brushing9.3 (7.9–10.6)Circular11.9 (11.7–12.0)
Second brushing12.4 (12.0–12.9)Circular12.5 (12.4–12.6)
Sibling 211.1 (10.6–11.6)Circular17.0 (16.8–17.3)
Sibling 3
11.6 (11.1–12.0)
Circular
13.5 (13.2–13.8)

Data are expressed as mean (95% confidence intervals). Normal data range for age from our laboratory: ciliary beat frequency 12.9 Hz ± 2.3 SD, central microtubule defects 0.0–1.2 (expressed as 5th–95th percentiles) (6).

. However, the beat pattern was abnormal in all cases. Instead of beating in a forward and backward planar motion, all the cilia were seen to beat in a circular motion on all epithelial edges studied (Table 2). This is a beat pattern characteristic of a ciliary transposition defect (3). As with the eldest sibling's first biopsy, a classical transposition defect was not demonstrated at transmission electron microscopy in any of the children. Although the central microtubules were found to be absent in 12.5 to 17.0% of cilia on cross-sectional analysis of the fields of cilia studied (Table 2), a peripheral doublet with associated dynein arms was not transposed to the center of the axoneme (Figure 2). Other microtubule defects did not exceed 3.8% in any sibling and were well within the normal range for their age (6). Likewise, dynein arm defects did not exceed 3.2% in any sibling, again well within the normal range (6).

The results of the nasal ciliary biopsies in these three children support the diagnosis of primary ciliary dyskinesia due to a defect of the central microtubule pairs. All three siblings had a normal ciliary beat frequency with a circular beat pattern typical of a transposition defect. However, electron microscopic studies did not demonstrate a classical transposition defect. An absence of the central pair of microtubules was demonstrated but without the transposition of a peripheral doublet. We believe this is the first report of this ultrastructural phenotype causing a circular beat pattern.

It is of interest that the ciliary beat pattern in these siblings was circular. This is the beat pattern seen not only in cilia with a transposition defect but also in primary nodal cilia that are found on the ventral surface of the embryonic node. Primary nodal cilia have a 9+0 microtubular arrangement, that is, they lack a central microtubule pair (7). Thus, our findings suggest that the rotational beat pattern is associated with the defect of the central microtubule pair and not the transposition of a peripheral microtubule doublet to the center of the axoneme as seen in ciliary transposition defects.

The rotational beat pattern of primary nodal cilia that results in leftward nodal flow of extracellular fluid is thought to determine situs. Studies involving situs mutant (inversus viscerum) mice demonstrated that the mutants had immotile nodal cilia, due to a mutation in the left–right dynein (lrd) heavy chain gene, which resulted in a lack of nodal flow (8). This, in turn, resulted in random left side determination. In a recent study that allowed embryos to develop in a flow culture system in which artificial nodal flow was controlled, most embryos that had an artificial rightward nodal flow exhibited a reversal of left–right situs (9). Similarly, an artificial leftward or rightward nodal flow in an inversus viscerum/inversus viscerum mutant mouse embryos resulted in normal and reversed left–right patterning, respectively (9). The randomization of situs seen in patients with primary ciliary dyskinesia secondary to dynein arm defects may be due to immotile nodal cilia and therefore a lack of nodal flow. In patients with a defect of central microtubules, the ultrastructure, and thus the function, of nodal cilia would not be affected. This would explain the absence of situs inversus in patients with a ciliary transposition or central microtubular pair defect (3, 4).

The diagnosis of primary ciliary dyskinesia is supported in all three cases by the history and clinical findings. The three children reported here had chronic upper and lower respiratory tract symptoms that started early in life, and the eldest sibling had bronchiectasis demonstrated on the computed tomography scan. In addition, the third sibling, although a term infant, received oxygen therapy at birth, which is not an uncommon occurrence in children with primary ciliary dyskinesia (10). The eldest sibling also had a very low nasal nitric oxide of 11.6 ppb. This corresponds to the very low levels reported previously in primary ciliary dyskinesia (11). Unfortunately, the two younger siblings were too young for the measurement of nasal nitric oxide to be performed. Parental consanguinity supports an autosomal recessive inheritance.

Although microtubular defects have been described as an acquired defect in epithelial damage due to, for example, chronic infection and irritant exposure (12, 13), this is unlikely in these siblings for several reasons. First, the same defect is demonstrated in all three children, in conjunction with the history and clinical findings. The history in the youngest sibling of respiratory difficulties from birth, without evidence of infection or insult, suggests that an acquired defect is unlikely, as does the persistence of the ultrastructural abnormalities in the eldest sibling after the initiation of treatment. The epithelium from the eldest sibling's second brush biopsy and from the two younger siblings was healthy, as determined by assessing epithelial integrity (Table 1). Furthermore, in acquired defects, in addition to epithelial disruption, heterogeneous microtubular defects and compound cilia are usually demonstrated (14). The percentage of central microtubule defects seen in these cases was significantly greater than the normal range for their age group seen in our laboratory (6) or those described in secondary defects (12, 13). The percentage of defects seen here is also similar to that seen in our cohort of patients with transposition defects (14.2% [95% confidence interval: 6.8–21.6]) (3). No reported case of primary ciliary dyskinesia due to transposition has demonstrated the ultrastructural defect in every cilium when a cross-sectional field is studied (3, 4). Even after ciliogenesis in culture, which has been shown to significantly reduce secondary defects, central pairs were absent in 21% of cilia in six patients with primary ciliary dyskinesia compared with 15% in the initial biopsy (15). However, it is not clear if this defect was a defect of absent central pairs or of transposition (16).

It is of interest that the circular ciliary beat pattern was observed in all the ciliated edges examined in the patients reported in this study and in patients with a transposition defect. In the transposition defect, it has been noted that the peripheral microtubular doublet can move into the center of the axoneme in the middle or even distal aspect of the cilium (4). Thus, in this complex, genetically heterogeneous disease, the defect may not occur at the same site in each cilium. The defect may be seen in only a certain percentage of each epithelial section, with the defect occurring in the remainder of the cilia above the plane examined. This may explain the reason for the ultrastructural defect being observed in less than a quarter of cilia in the transposition defect and in these siblings with absent central pairs.

In summary, we report a diagnosis of primary ciliary dyskinesia in three siblings due to a defect of the central microtubular pair that results in a circular beat pattern. We would propose the name, central microtubular agenesis, to describe the ultrastructure of this ciliary dyskinesia.

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Correspondence and requests for reprints should be addressed to Chris O'Callaghan, F.R.C.P., F.R.C.P.C.H., D.M., Ph.D., Division of Child Health, Institute of Lung Health, Department of Infection, Immunity, and Inflammation, University of Leicester, Robert Kilpatrick Clinical Sciences Building, PO Box 65, Leicester Royal Infirmary, Leicester LE2 7LX, UK. E-mail:

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