CiliarMove: new software for evaluating ciliary beat frequency helps find novel mutations by a Portuguese multidisciplinary team on primary ciliary dyskinesia

Sampaio, Pedro; Silva, Mónica Ferro da; Vale, Inês; Roxo-Rosa, Mónica; Pinto, Andreia; Constant, Carolina; Pereira, Luí­sa Álvares; Quintão, Carla; Lopes, Susana S.

doi:10.1183/23120541.00792-2020

Cited by 22 publications

(29 citation statements)

References 49 publications

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“…Whereas Sampaio et al (2014) and Tavares et al (2017) have extensively described the rotational and wavy fashion beat of monocilia of the LRO[16,18]. These motile monocilia are essential to generate flow for the determination of left-right asymmetry[16,18,41]. We confirmed the localization and distribution of cilia in the wild-type (WT) LRO and OP of zebrafish by immunofluorescence (IF).…”

supporting

confidence: 72%

“…[16,18]. These motile monocilia are essential to generate flow for the determination of left-right asymmetry [16,18,41]. We confirmed the localization and distribution of cilia in the wild-type (WT) LRO and OP of zebrafish by immunofluorescence (IF).…”

Section: Zebrafish Motile Ciliasupporting

confidence: 61%

“…Next, we evaluated the cilia beat frequency for both organs, so that, in the future, researchers can compare it with zebrafish disease models for PCD. CBF in the LRO ranges from 15 to 50 Hz was evaluated by CiliarMove [41]. This software creates a heatmap for CBF that allowed us to unequivocally detect in a very visual way different cilia in the same focal plane beating differently as depicted by the blue, green and yellow ciliary colour codes (Figure 3A').…”

Section: Zebrafish Motile Ciliamentioning

confidence: 99%

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Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

Pinto

Rasteiro

Bota

et al. 2021

IJMS

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Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left–right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.

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supporting

confidence: 72%

Section: Zebrafish Motile Ciliasupporting

confidence: 61%

Section: Zebrafish Motile Ciliamentioning

confidence: 99%

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Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

Pinto

Rasteiro

Bota

et al. 2021

IJMS

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“…Six separate ciliated epithelium strips (five sideway edges and one from above) with mucus-free regions from the subject were analyzed. The ciliary beat frequency was calculated using the validated automated open-source software CiliarMove ( Sampaio et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Novel RSPH4A Variants Associated With Primary Ciliary Dyskinesia–Related Infertility in Three Chinese Families

Wang

Wang²,

Yang³

et al. 2022

Front. Genet.

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Background: The radial spoke head component 4A (RSPH4A) is involved in the assembly of radial spokes, which is essential for motile cilia function. Asthenoteratozoospermia in primary ciliary dyskinesia (PCD) related to RSPH4A variants has not been reported.Materials and Methods:RSPH4A variants were identified and validated using whole-exome and Sanger sequencing in three unrelated Chinese families. High-speed video microscopy analysis (HSVA) was performed to measure the beating frequency and pattern of nasal cilia of the patients and healthy control. Papanicolaou staining and computer-aided sperm analysis were performed to analyze the morphology and motility of the sperm in patient 1. Immunofluorescence was adopted to confirm the structure deficiency of sperm and nasal cilia.Results: Patient 1 from family 1 is a 22-year-old unmarried male presented with bronchiectasis. Semen analysis and sperm Papanicolaou staining confirmed asthenoteratozoospermia. Novel compound heterozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.1774_1775del, p.(Leu592Aspfs*5) were detected in this patient. Patients 2 and 3 are from two unrelated consanguineous families; they are both females and exhibited bronchiectasis and infertility. Two homozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.351dupT, p.(Pro118Serfs*2) were detected, respectively. HSVA showed that most of the cilia in patients 1 and 3 were with abnormal rotational movement. The absence of RSPH4A and RSPH1 in patient 1’s sperm and patient 3’s respiratory cilia was indicated by immunofluorescence. Patient 2 died of pulmonary infection and respiratory failure at the age of 35 during follow-up.Conclusion: Dysfunctional sperm flagellum and motile cilia in the respiratory tract and the fallopian tube were found in patients with RSPH4A variants. Our study enriches the genetic spectrum and clinical phenotypes of RSPH4A variants in PCD, and c.2T>C, p.(Met1Thr) detected in our patients may be a hotspot RSPH4A variant in Chinese.

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“…12 The ciliary beat frequency was calculated using CiliarMove, a validated software. 13 A mix of normal cilia beating and stiff beating was observed and the beat frequency of the patient with the OFD1 variant was higher than that of the healthy control (13.02 ± 3.61 Hz vs 9.52 ± 0.52 Hz) (Figure 3A and Supplementary video 1 and 2). The normal ultrastructure of the axoneme was confirmed in TEM.…”

Section: Detailed Examination Of the Respiratory Cilia And The Sperm ...mentioning

confidence: 96%

Identification of a Novel OFD1 Variant in a Patient with Primary Ciliary Dyskinesia

Yang

Cheng

Yang

et al. 2022

PGPM

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Background: OFD1 encodes a protein with 1012 amino acids, which is a component of basal bodies and centrioles, essential for cilia biogenesis. OFD1 was reported to be associated with X-chromosome linked dysmorphology syndrome in early studies and recent studies reported a few cases with primary ciliary dyskinesia (PCD) caused by OFD1 deficiency. Case Presentation: We report a 31-year-old man who suffered from recurrent respiratory infections with typical manifestations of primary ciliary dyskinesia. In addition to respiratory manifestations, the patient also had situs inversus, obesity, gastroesophageal reflux, and hearing impairment. Clubbing fingers and mild streblomicrodactyly were also observed. Examination Result: We performed whole-exome sequencing to identify a novel variant c.2795delA:p.(Lys932Argfs*3) in OFD1. The hemizygous variant was predicted to be likely pathogenic by bioinformatic analysis software and ACMG guideline. High-speed video microscopy (HSVM), transmission electron microscopy (TEM), and immunofluorescence were performed to analyze the respiratory cilia. A high beating frequency and a stiff beating pattern were observed under HSVM, while there were no significant abnormalities in TEM and immunofluorescence. The sperm flagella examinations were also generally normal. Conclusion:Our study identified a novel frameshift variant in OFD1 causing PCD, enriched the genetic spectrum of OFD1 variants, and verified that OFD1 mutation can lead to only a PCD characteristic phenotype, while other OFD1-associated syndromic symptoms such as dysmorphic features and renal symptoms were not present.

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CiliarMove: new software for evaluating ciliary beat frequency helps find novel mutations by a Portuguese multidisciplinary team on primary ciliary dyskinesia

Cited by 22 publications

References 49 publications

Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

Novel RSPH4A Variants Associated With Primary Ciliary Dyskinesia–Related Infertility in Three Chinese Families

Identification of a Novel OFD1 Variant in a Patient with Primary Ciliary Dyskinesia

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