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. 2018 Feb 2;9:23.
doi: 10.3389/fgene.2018.00023. eCollection 2018.

Compound Heterozygous Variants in the Coiled-Coil Domain Containing 40 Gene in a Chinese Family With Primary Ciliary Dyskinesia Cause Extreme Phenotypic Diversity in Cilia Ultrastructure

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Compound Heterozygous Variants in the Coiled-Coil Domain Containing 40 Gene in a Chinese Family With Primary Ciliary Dyskinesia Cause Extreme Phenotypic Diversity in Cilia Ultrastructure

Lin Yang et al. Front Genet. .
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Abstract

Purpose: Primary ciliary dyskinesia (PCD) is a rare genetic disorder manifested with recurrent infections of respiratory tract and infertility. Mutations in more than 20 genes including the Coiled-Coil Domain Containing 40 (CCDC40) gene are associated with PCD. A Chinese proband with a clinical diagnosis of PCD was analyzed for mutations in these genes to identify the genetic basis of the disease in the family. The proband showed altered mucociliary clearance of the airways, various degree of hyperemia and edema of the mucous membrane, left/right body asymmetry, infertility and ultrastructural abnormality of cilia in both sperm and bronchioles. Methods: The DNA from the proband was analyzed for genetic variation in a subset of genes known to cause PCD using targeted next generation sequencing in order to understand the molecular and genetic basis of the PCD in present family. The result of targeted next generation sequencing has been validated by Sanger sequencing and q-PCR. Results: Targeted next-generation sequencing identified two novel mutations (c.1259delA and EX17_20 deletion) in CCDC40 gene that causes abnormal CCDC40 mRNA expression. These two novel variants cause disorganization of axoneme filaments, which resulted in reduction of sperm motility and phenotypic diversity in ultrastructure of cilia in the proband. Conclusion: These findings highlight the significance of the mutations in CCDC40 as novel candidates for genetic testing in PCD patients as well as the key role of ICSI treatment for the families affected by this ciliary dysmotility. Our findings showed that our work enriched the performance of cilia ultrastructure which were not previously reported in PCD patients.

Keywords: CCDC40; PCD; intracytoplasmic sperm injection; mRNA expression; targeted next-generation sequencing.

Figures

FIGURE 1
FIGURE 1
Pedigree structure of the family with Primary ciliary dyskinesia. Family members with Primary ciliary dyskinesia are indicated with shading. Squares and circles denoted males and females, respectively. Triangles denoted fetuses when II-3 was artificially aborted fetus, and II-4 was spontaneous aborted fetus. Individuals labeled with a slash were deceased. Individuals undetected or without gene mutation are indicated with no symbols. Roman numerals indicate generations. Arrow indicates the proband (II-5). Family members detected with CCDC40 gene mutation (c.1259delA) are indicated with stars (aaa). Family members detected with CCDC40 gene deletion (EX17_20del) are indicated by “four angled star” ([scale=.5]img001).
FIGURE 2
FIGURE 2
Radiological examination of Proband and Proband’s Sister. The radiological examination of Proband (II-5), (A) CT showed situs inversus. (B) Coronal view of paranasal sinus MRI shows bilateral maxillary sinusitis and ethmoid sinusitis. (C,D) HRCT of chest shows bronchitis and bronchiolitis. Bronchiectasia involves in left lobes. Centrilobular nodules and tree-in-bud sign distribute diffusely in both lobes.The radiological examination of Proband’s sister (II-1), (E) CT showed situs inversus, (F) Transverse view of sinus MRI showed bilateral maxillary sinusitis, (G,H) HRCT of chest showed bronchitis and bronchiolitis, ring-shaped or ductal opacities bronchiectasia involves in both lobes. Secretion obstruction can be seen in some saccular bronchiectasis in the upper left lobes.
FIGURE 3
FIGURE 3
Bronchoscopy examination. Proband‘s (II-5) Bronchoscopy examination: (A) mucosal biopsy were performed on the proband’s right middle bronchus, (B) right lower lobe basal section of the bronchus, (C) carina of trachea. A small amount of purulent secretion is secreted by the tracheal carina. Bronchoscopy examination of the proband’s elder sister (II-1): (D) mucosal biopsy were performed on the elder sister’s right middle bronchus, (E) right lower lobe basal section of the bronchus, (F) carina of trachea. A large number of purulent secretion are secreted by the tracheal carina.
FIGURE 4
FIGURE 4
Bronchial ciliary electron microscopy of the proband (II-5). Proximal bronchial ciliary electron microscopy of the proband, (A) several microtubules in vertical section, microtubules run through from the top to the base, their relative location stay the same, (B) transverse section of the cilia, part of the cilia plasma membrane fusion to form composite cilia, (C) cilia in transverse section, red arrow:normal 9+2 structure, the radial spokes structures and DAs are faintly recognizable; yellow arrow: 9+0 structure, the position of peripheral microtubules are shifted, central microtubules disappear; blue and orange arrow:9+0 structure, the position of peripheral microtubules are completely irregular, central microtubules disappear, irregular shape of cross section; green arrow: composite cilia, irregular shape of cross section, one pair of central microtubules and several peripheral microtubules, complete confusion of position. Total chaos of radial spokes and DAs structures. (D) The distal bronchial mucosa cilia electron microscopy of the proband; red arrow: normal 9+2 structure, the radial spokes structures and DAs are recognizable; yellow arrow: the number and position of peripheral microtubules are normal, the central microtubules shifted; blue arrow: the position of one peripheral microtubule and central microtubule are shifted. (E) The middle section of bronchial mucosa cilia electron microscopy of the proband; red arrow: normal 9+2 structure, the radial spokes structures and DAs are faintly recognizable; yellow arrow: 8+0 structure, the number of peripheral microtubules are reduced, and position are completely irregular, central microtubules disappear; blue arrow: 8+2 structure, the number of peripheral microtubules are reduced, position of microtubules are shifted; orange arrow: 9+2 structure, the position of peripheral microtubules are shifted; green arrow: 9+2 structure, the position of all microtubules are shifted. (F) Bronchial ciliary electron microscopy of the proband’s sister showed more cells with oval synapses (Indicated by the red circle).
FIGURE 5
FIGURE 5
Transmission electron microscope (TEM) analyses of CCDC40-mutant sperm cilia ultrastructural abnormality from proband. (A,B) Optical microscope of Diff-quick stained sperm from the PCD patient indicated the curly tail of sperm. (C) Vertical section of a sperm, the heads are normal while the flagellum is flexural and encircled with the plasma membrane, notice the disarrangements of the mitochondria. (D,E) Cross section of mid piece region of flagellum with normal mitochondria packing at 98,000× magnification; morphology of outer dense fibers ODF(s) appeared normal, 1–3 ODFs are retained but mislocalised; absent central pairs with eccentric number and displacement of outer doublets; the radial spokes structures are reduced or absent; (D) also showed faintly visible derangements of IDA and ODA; (E) also showed absence of IDA and ODA. (F,G) Cross section of principal piece region of flagellum; abnormal quantities and disorganization of ODFs arrangement; central microtubule pair get lost, decreased in number with perturbed peripheral microtubular doublets structure and radial spokes, dynein arms are beyond recognition. Excess fibrous sheaths are observed. (H,I) Two section of the same flagellum move toward in the opposite direction, surrounded by the same outer membrane. (H) Showed cross section of mid piece (left) and principal piece (right), the perturbations of axoneme structure get more and more worse from the proximal to distal. (I) Showed that both the mid piece region of flagellum showed obvious structural abnormality, note the absence of a truncated lateral column (arrow), decreased central pair and acentric microtubular central pairs.
FIGURE 6
FIGURE 6
Sanger Sequencing, the qPCR results and the analysis of CCDC40 mRNA relative expression (n = 3). (A) Sanger sequencing analysis of CCDC40 mutation in the family. The deletion (c.1259delA) was observed in the mother (I:2), and inherited to both daughter (II:1) and son (II:5). The other deletion (EX 17_20del) was observed in the father (I:1), and inherited to both daughter (II:1) and son (II:5). (B) A large deletion (EX17_20del) was confirmed by qPCR in the family members (I:1, II:1 and II:5). (C) Stands for different statistical differences: P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and indicate the significant differences between groups. UD means “Undetermined,” when no data output can be detected from qPCR, it would reminder “undetermined.” However, here the “UD” should represent that mRNA expression was very low, which may relate to the RNA initial concentration of blood. “N = 3” means each blood sample was used to establish three qPCR amplification system, respectively. The CCDC40 mRNA relative expression of both proband (II:5) and his sister (II:1), who carry two kinds of mutation, was apparently lower that the expression of their parents with normal phenotype.
FIGURE 7
FIGURE 7
Working dish for ICSI procedure.

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