Abstract
Purpose
Usher syndrome is a ciliopathy involving photoreceptors and cochlear hair cells (sensory cilia): since sensory and motor ciliopathies can overlap, we analysed the respiratory cilia (motile) in 17 patients affected by Usher syndrome and 18 healthy control subject.
Patients and methods
We studied the mucociliary transport time with the saccharine test, ciliary motility and ultrastructure of respiratory cilia obtained by nasal brushing; we also recorded the classical respiratory function values by spirometry.
Results
All enrolled subjects showed normal respiratory function values. The mean mucociliary transport time with saccharine was 22.33 ± 17.96 min, which is in the range of normal values. The mean ciliary beat frequency of all subjects was 8.81 ± 2.18 Hz, which is a value approaching the lower physiological limit. None of the classical ciliary alterations characterizing the “ciliary primary dyskinesia” was detected, although two patients showed alterations in number and arrangement of peripheral microtubules and one patient had abnormal ciliary roots.
Conclusions
Respiratory cilia in Usher patients don’t seem to have evident ultrastructural alterations, as expected, but the fact that the ciliary motility appeared slightly reduced could emphasize that a rigid distinction between sensory and motor ciliopathies may not reflect what really occurs.
1
Introduction
Cilia carry out different functions and are broadly divided into two types: “motile” cilia, such as respiratory cilia, and “primary” cilia, generally non-motile, which transduce extracellular signals to the interior of the cell. However, there are some exceptions in this simplified classification. The two classes of cilia collectively perform a wide variety of functions sometimes overlapping, encompassing fluid movement and sensory perception .
Motile cilia, characterized by a coordinate rhythmic wave-like motion, are mostly found in the respiratory tract, where 100–200 cilia cluster on the epithelial cell surface; the ciliary beat frequency in the airway ranges approximately from 10 to 12 Hz in normal conditions to propel the mucus film . They also line the Eustachian tubes and sinuses, the ependymal cells of the brain ventricles as well as the fallopian tubes of the female reproductive system. Functions of motile cilia include clearing airways, circulating cerebrospinal fluid and facilitating fertilization . In general, they display a 9 + 2 arrangement, consisting of nine peripheral microtubule doublets surrounding two central single microtubules .
The internal structure of primary cilia differs noticeably from that of epithelial motile cilia; they lack the main elements involved in motility, including central pairs of microtubules (9 + 0 pattern), and are devoid of dynein arms . Primary cilia are covered with a specialized membrane containing a distinct subset of receptors and are studded with a variety of receptors and ion channels proteins; these features have been shown to represent primary cilia as key structures in the transduction of a range of molecular signals to coordinate and regulate interactions between cells and their environment .
More than 200 proteins are involved in the synthesis and assembly of cilia. The synthesis of structural and functional cilia components occurs in the cytoplasm and a specialized system of intraflagellar transport particles moves these elements toward the axonemal tip (anterograde transport) and away from it (retrograde transport) . The intraflagellar transport is not only necessary for transport of axonemal components but it is also important for the sensory activity of cilia .
As the cilium contains several hundreds of proteins in addition to this basic structure, the inactivation of any one of them may be sufficient to impair cilium function(s), resulting in a variability of clinical manifestations and different phenotypic severity. With cilia being present in almost all organs of the human body, their dysfunctions underlie a wide range of disorders and developmental abnormalities, collectively termed “ciliopathies”, which can stem from defects in motor and/or sensory cilia .
Among ciliopathies, Usher syndrome (USH) is part of a clinically and genetically heterogeneous group, comprising at least three clinical entities, USH1, USH2, USH3: all manifest combined sensorineural hearing loss with varying age of onset and visual loss due to a progressive retinal degeneration termed “retinitis pigmentosa” . USH type 1 is the most severe form of the Usher syndrome: this form accounts for 30%–40% of Usher syndrome cases and is characterized by the presence of congenital, profound deafness, vestibular dysfunction and prepuberal onset of retinitis pigmentosa. The USH type 2 is less severe than type 1, is the most frequent type accounting for over a half of all cases, and is characterized by congenital moderate-to-severe deafness and a considerable variation in the clinical appearance of the visual symptoms. The USH type 3 is less common, has a prevalence of 2%–4% and is characterized by variable onset of progressive hearing loss and of retinitis pigmentosa . At least 9 genes associated with different types of USH have been identified, most of which encode motor and structural proteins localized to the photoreceptor-connecting cilia or to the common synaptic and periciliary areas of the ear and eye .
Knowing that sensory and motor ciliopathies can overlap , in the attempt to better elucidate the relationship between alterations in photoreceptors and cochlear hair cells (sensory cilia) and the respiratory cilia (motor cilia), we have undertaken a prospective recruitment of Usher’s patients to study their respiratory mucociliary clearance and ciliary ultrastructure. To our knowledge, similar studies on a significant group of Usher patients have not been performed before.
2
Methods
2.1
Sample selection
We enrolled 17 patients (9 M, 8 F median age: 32.7 ± 11.97 years) affected by Usher syndrome (2 USH1, USH1B and USH1D and 15 USH2) and 18 healthy control subjects (7 M, 9 F) of similar age (32 ± 8.43 years), all recruited from the Audiology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy from February 2006 to November 2008.
Patients underwent a full clinical examination, with particular regard to othorhinolaryngological and respiratory assessment and to history of recurrent upper and lower airway infections.
A spirometry was performed to evaluate their respiratory function. Mucociliary transport was studied by saccharine test and the ciliary motility was examined on nasal epithelium obtained by brushing. An additional nasal brushing was performed to study ciliary ultrastructure.
None of the patients had reported acute respiratory tract diseases in the month preceding the study; during the same time none of the patients took antibiotics.
The research followed the tenets of the Declaration of Helsinki.
Patients signed an informed consent and the protocol was approved by the Ethical Committee of the Ospedale Maggiore Policlinico of Milan, Italy (25/11/2005).
2.2
Molecular genetic analysis
Genomic DNA was extracted from peripheral blood of each Usher patient according to standard protocols and PCR amplified. The coding exons and flanking intron sequences of MYO7A, PCDH15, CDH23, USH1C and USH1G were analysed by direct sequencing of 189 amplicons as described in details by Roux et al. 2006 .
2.3
Spirometry
Spirometric parameters (FEV1, FVC, Tiffeneau index) were measured according to American Thoracic Society criteria . An FEV1 > 85% of the predicted value was defined as normal. Each test was performed three times and the best performance was selected.
2.4
Saccharine test
The mucociliary transport time was measured with a saccharine test . Using bayonet forceps, a saccharine granule (1 mm diameter) was inserted into the nasal speculum and placed gently onto the superior surface of the inferior turbinate, about 1.5 cm inside the nostril. The time taken for the subject to perceive a sweet taste, representing transport of saccharine from the anterior part of the nose to the oropharynx, was recorded. Usually, normal subjects perceive a sweet taste within 30 min; if no taste was experienced by 60 min, the test was stopped. The test was carried out in a comfortable and quiet room with the patient sitting on an examination chair at 90° and head held upright. During the test, the patient was requested not to eat, drink, talk or sneeze. All saccharine tests were undertaken by the same researcher.
2.5
Ciliary motility study
Samples of ciliated epithelium were obtained from the deep surface of the inferior nasal turbinate under direct inspection using a Cytology Brush (Olympus, dia 1 mm). Nasal cilia were chosen for this study because multiple specimens are obtained simply and with little discomfort for the subjects; in addition, it has been previously demonstrated that they have similar functional and structural characteristics to bronchial cilia . After brushing, adherent epithelium was dislodged by gently shaking the sample into a plastic tube containing a nutrient medium (Medium 199). Several drops of the suspension containing multiple fragments of mucosa were placed in a chamber formed between a microscope slide and a coverslip sealed at the edges with vaseline. The slide was placed on a heated microscope stage (22 °C) and ciliary beat frequency (CBF) was measured. Observations were made under an Olympus BH-2 optical microscope equipped with Normarsky interferential contrast. The microscope was connected to a monitor. The monitor was equipped with a visual and auditory marker that pulses at known adjustable frequencies: CBF was calculated by synchronising ciliary motion of the sample with marker pulse frequency and expressed in Hz . Only large groups of cells in continuous strips were chosen randomly for assessment and an average of ten random areas were considered. CBF measurements were made within 30 min from the brushing sample being taken and were always performed by the same observer. The mean value of CBF was calculated. Observations were performed at a final magnification of 400 × and 1000 ×. Besides CBF, we considered the number of ciliated cells and the quality of ciliary movement. Normal ciliary beat frequency usually ranges from 10 to 12 Hz .
2.6
Transmission Electron microscopy (TEM)
Samples of respiratory epithelium were obtained by nasal brushing and immediately fixed in 2.5% cacodylate-buffered glutaraldehyde, postfixed in buffered 1% osmium tetroxide, dehydrated through a graded series of ethanol, cleared in propylene-oxide and embedded in Epoxy resin (Araldite). Semi-thin sections 1 μm thick, cut with glass knives on an LKB V Ultrotome and stained with 1% toluidine blue, were examined with the light microscope for general evaluation of morphology. Ultrathin sections from selected areas were cut with a diamond knife using the same ultramicrotome, retrieved onto copper grids, double-stained with uranyl acetate and lead citrate and examined at 100 kV with a Philips 208 S transmission electron microscope.
At least 50 cross sections of different cilia from different cells were observed in each specimen to study the axonemal structure. Only full cross-sectioned cilia were evaluated, excluding those near the base or tip. Electron micrographs were taken at a magnification of × 50,000 to determine the orientation of the cilia and at a magnification of × 110,000 to study the axonemal pattern. Dynein arms and microtubules were counted and the organization of the axoneme, the presence of radial spokes, spoke heads and central sheaths were evaluated. The incidence of abnormal cilia was expressed as a percentage.
From each specimen, ciliary orientation was investigated observing at least 10 different cells. The ciliary axis was determined drawing a line through the central microtubular pair of each cilium. At least 10 suitable ciliary cross sections per cell were studied: the angles between the ciliary axis and a standard reference line were measured and the standard deviation of the angles per cell was calculated. Finally, the mean standard deviation of the examined cells of the same specimen was calculated.
2.7
Statistical evaluation
We performed a Student t-test to compare the mucociliary transport time with saccharine test and the ciliary beat frequency in Usher and healthy control subjects. A p < 0.05 has been considered statistically significant, and p < 0.01 highly significant.
2
Methods
2.1
Sample selection
We enrolled 17 patients (9 M, 8 F median age: 32.7 ± 11.97 years) affected by Usher syndrome (2 USH1, USH1B and USH1D and 15 USH2) and 18 healthy control subjects (7 M, 9 F) of similar age (32 ± 8.43 years), all recruited from the Audiology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy from February 2006 to November 2008.
Patients underwent a full clinical examination, with particular regard to othorhinolaryngological and respiratory assessment and to history of recurrent upper and lower airway infections.
A spirometry was performed to evaluate their respiratory function. Mucociliary transport was studied by saccharine test and the ciliary motility was examined on nasal epithelium obtained by brushing. An additional nasal brushing was performed to study ciliary ultrastructure.
None of the patients had reported acute respiratory tract diseases in the month preceding the study; during the same time none of the patients took antibiotics.
The research followed the tenets of the Declaration of Helsinki.
Patients signed an informed consent and the protocol was approved by the Ethical Committee of the Ospedale Maggiore Policlinico of Milan, Italy (25/11/2005).
2.2
Molecular genetic analysis
Genomic DNA was extracted from peripheral blood of each Usher patient according to standard protocols and PCR amplified. The coding exons and flanking intron sequences of MYO7A, PCDH15, CDH23, USH1C and USH1G were analysed by direct sequencing of 189 amplicons as described in details by Roux et al. 2006 .
2.3
Spirometry
Spirometric parameters (FEV1, FVC, Tiffeneau index) were measured according to American Thoracic Society criteria . An FEV1 > 85% of the predicted value was defined as normal. Each test was performed three times and the best performance was selected.
2.4
Saccharine test
The mucociliary transport time was measured with a saccharine test . Using bayonet forceps, a saccharine granule (1 mm diameter) was inserted into the nasal speculum and placed gently onto the superior surface of the inferior turbinate, about 1.5 cm inside the nostril. The time taken for the subject to perceive a sweet taste, representing transport of saccharine from the anterior part of the nose to the oropharynx, was recorded. Usually, normal subjects perceive a sweet taste within 30 min; if no taste was experienced by 60 min, the test was stopped. The test was carried out in a comfortable and quiet room with the patient sitting on an examination chair at 90° and head held upright. During the test, the patient was requested not to eat, drink, talk or sneeze. All saccharine tests were undertaken by the same researcher.
2.5
Ciliary motility study
Samples of ciliated epithelium were obtained from the deep surface of the inferior nasal turbinate under direct inspection using a Cytology Brush (Olympus, dia 1 mm). Nasal cilia were chosen for this study because multiple specimens are obtained simply and with little discomfort for the subjects; in addition, it has been previously demonstrated that they have similar functional and structural characteristics to bronchial cilia . After brushing, adherent epithelium was dislodged by gently shaking the sample into a plastic tube containing a nutrient medium (Medium 199). Several drops of the suspension containing multiple fragments of mucosa were placed in a chamber formed between a microscope slide and a coverslip sealed at the edges with vaseline. The slide was placed on a heated microscope stage (22 °C) and ciliary beat frequency (CBF) was measured. Observations were made under an Olympus BH-2 optical microscope equipped with Normarsky interferential contrast. The microscope was connected to a monitor. The monitor was equipped with a visual and auditory marker that pulses at known adjustable frequencies: CBF was calculated by synchronising ciliary motion of the sample with marker pulse frequency and expressed in Hz . Only large groups of cells in continuous strips were chosen randomly for assessment and an average of ten random areas were considered. CBF measurements were made within 30 min from the brushing sample being taken and were always performed by the same observer. The mean value of CBF was calculated. Observations were performed at a final magnification of 400 × and 1000 ×. Besides CBF, we considered the number of ciliated cells and the quality of ciliary movement. Normal ciliary beat frequency usually ranges from 10 to 12 Hz .
2.6
Transmission Electron microscopy (TEM)
Samples of respiratory epithelium were obtained by nasal brushing and immediately fixed in 2.5% cacodylate-buffered glutaraldehyde, postfixed in buffered 1% osmium tetroxide, dehydrated through a graded series of ethanol, cleared in propylene-oxide and embedded in Epoxy resin (Araldite). Semi-thin sections 1 μm thick, cut with glass knives on an LKB V Ultrotome and stained with 1% toluidine blue, were examined with the light microscope for general evaluation of morphology. Ultrathin sections from selected areas were cut with a diamond knife using the same ultramicrotome, retrieved onto copper grids, double-stained with uranyl acetate and lead citrate and examined at 100 kV with a Philips 208 S transmission electron microscope.
At least 50 cross sections of different cilia from different cells were observed in each specimen to study the axonemal structure. Only full cross-sectioned cilia were evaluated, excluding those near the base or tip. Electron micrographs were taken at a magnification of × 50,000 to determine the orientation of the cilia and at a magnification of × 110,000 to study the axonemal pattern. Dynein arms and microtubules were counted and the organization of the axoneme, the presence of radial spokes, spoke heads and central sheaths were evaluated. The incidence of abnormal cilia was expressed as a percentage.
From each specimen, ciliary orientation was investigated observing at least 10 different cells. The ciliary axis was determined drawing a line through the central microtubular pair of each cilium. At least 10 suitable ciliary cross sections per cell were studied: the angles between the ciliary axis and a standard reference line were measured and the standard deviation of the angles per cell was calculated. Finally, the mean standard deviation of the examined cells of the same specimen was calculated.
2.7
Statistical evaluation
We performed a Student t-test to compare the mucociliary transport time with saccharine test and the ciliary beat frequency in Usher and healthy control subjects. A p < 0.05 has been considered statistically significant, and p < 0.01 highly significant.
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