PMC:4157146 / 19692-40005 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4157146","sourcedb":"PMC","sourceid":"4157146","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4157146","text":"Results\n\nIdentification of CCDC151 Mutations through High-Throughput Autozygosity Mapping and Sequencing\nWe used a high-throughput next-generation sequencing (NGS) approach to identify PCD-causing mutations in affected individuals that were clinically diagnosed with PCD caused by deficiency of the axonemal ODAs. The NGS pipeline consisted of either whole-exome sequencing or a targeted panel-based resequencing of selected candidate genes, performed in two separate cohorts of individuals with PCD as previously reported.11,42 NGS data were processed through standard quality controls, and sequence reads were aligned back to the genome and annotated for DNA variants, which were then filtered according to a rare recessive disease model11,42 (Tables S2 and S3). This excluded genes that did not have at least one homozygous or two heterozygous changes that were either previously unreported or occurring with an estimated frequency of less than 0.01 in publically available human exome databases (1000 Genomes, NHLBI EVS, dbSNP139). All variants except those predicted to produce a nonsynonymous or splice-site substitution, or an indel, were then removed. For the cases processed through exome sequencing, a filter was also applied to remove variants that were not in chromosomal regions of interest highlighted by autozygosity mapping.\nIn both the resequencing panel and exome-sequence analysis, all variants meeting the filtering criteria were finally examined to identify those present in genes predicted to have motile cilia function. From gene panel analysis that was conducted on 70 affected individuals, this revealed a homozygous single-base substitution in CCDC151 (RefSeq NM_145045.4), c.925G\u003eT, predicting a premature termination of translation p.Glu309∗, in a Bedouin-Arabic individual (UCL-65 II:8). The NGS sequence filtering steps taken to reveal this predicted loss-of-function variant as the likely disease cause are shown in Table S2. Individual UCL-65 II:8 also carries a single heterozygous variant in DNAH2 (rs7601298) and a homozygous variant in DNAH3 (rs138753702), which are predicted to be damaging to protein function, but these genes encode inner arm dyneins.50 These and a homozygous CCDC40 variant (rs10693712), described fully in Table S2, were not considered a possible cause of outer dynein arm loss and furthermore were excluded as causal by segregation analysis. The other variant meeting the filtering criteria was CCDC151 c.925G\u003eT (p.Glu309∗), which was also the only biallelic stop-gain mutation detected.\nIn the exome analysis that was conducted on 28 affected individuals, an autozygosity linkage mapping approach was employed in consanguineous families as an extra filter to analyze sequence variants in a narrowed set of chromosomal regions of interest. Genome-wide SNP genotyping (Affymetrix human SNP Array v.6.0) identified regions of homozygosity unique to the affected sibling in one UK-based consanguineous family of Pakistani origin (71154). We compared these data to SNP mapping data that could be derived from exome sequencing, to assess its utility for genetic mapping. We found that the information derived from both data sets was almost identical, identifying 15 autozygous regions of interest totalling 177 Mb unique to the affected sibling 71154 II:2, including a large autozygous region on chromosome 19p13 containing CCDC151 (Figure S1). A homozygous CCDC151 single-base substitution c.1256C\u003eA was identified in individual 71154 II:2 on the basis of prioritizing these linked regions, predicting a premature termination of translation p.Ser419∗ (Figures 1A and 1B). The NGS sequence filtering steps applied in family 71154 are summarized in Table S3.\nFamilial segregation analysis performed in available family members showed the CCDC151 variant inheritance pattern to be consistent for an autosomal-recessive disease in both families, including in a second affected sibling (UCL-65 II:7) (Figure 1A). This approach to derive autozygosity linkage mapping data from polymorphic markers and then focus on linkage-positive regions to identify rare disease-causing variants is powerful for exome-based gene discovery in consanguineous families, potentially removing the need for the extra cost of SNP array analysis in such families. Exome-sequencing-based linkage mapping has previously been tested for PCD51 and has proven successful for other genetic conditions.52\nCCDC151 is the vertebrate ortholog of Chlamydomonas reinhardtii ODA10, which was recently shown to be required for ODA assembly in these ciliated algae.53 We therefore considered CCDC151 a reasonable PCD candidate gene and proceeded to screen for CCDC151 mutations by Sanger sequencing in 150 additional affected individuals with ODA defects documented either by transmission electron microscopy (TEM) or by immunofluorescence analysis (IF). After PCR amplification and sequence analysis of all 13 exons, we identified CCDC151 mutations in individual OP-675, who carried the same homozygous nonsense mutation found in family UCL-65 (c.925G\u003eT [p.Glu309∗]). Familial segregation confirmed that this variant again segregated with the disease status in the wider family, including in another affected sibling, OP-1255 (Figure 1A). Neither of the two variants identified in this study, c.925G\u003eT (p.Glu309∗) or c.1256C\u003eA (p.Ser419∗), is present in the 1000 Genomes or Exome Variant Server databases.\nIn total, the mutational analysis detected CCDC151 loss-of-function nonsense mutations affecting five PCD individuals in three families. All the affected individuals displayed a clinical phenotype consistent with PCD including recurrent upper and lower airway disease with chronic respiratory symptoms and bronchiectasis, as well as nasal blockages, polyps, and otitis media. In all but one affected person, there was very early involvement with neonatal respiratory distress syndrome (Table S4). Four of the five affected individuals had laterality defects (Figure 1C, Table S4), with a congenital cardiac defect documented in individual OP-675 who had a ventricular septal defect (VSD). These clinical findings suggest that CCDC151 deficiency causes PCD and that CCDC151 function is required for correct laterality determination, which is consistent with the known role of ODA-generated ciliary motility in determining situs-specific morphogenesis.6\nPrevious studies supporting our human genetic data have shown that the Chlamydomonas CCDC151 ortholog ODA10 is a constituent of the flagella axoneme and is also present in the cell body.53 The null mutant oda10 Chlamydomonas strain lacks outer dynein arms, and ccdc151 deficiency in zebrafish knockdown morphants also causes a specific loss of ODAs.54 Human CCDC151 encodes a protein of 595 amino acids, and we used SMART to detect its domains, confirming that human CCDC151 is predicted to have three highly conserved coiled-coil domains. Coiled-coil domains are present in numerous proteins of diverse function and are recognized for their abundance in transcription factors involved in cell growth and proliferation and for their role in mediating interactions with other proteins.55 The CCDC151 mutations we identified in the PCD families are both predicted to cause premature protein truncations located within coiled-coil domains and would likely disrupt protein function (Figure 1D).\n\nccdc151 Is Mutated in the Zebrafish flanders Mutant, Leading to Ciliary Defects Including Laterality Defects\nThe evolutionarily conserved role of CCDC151 in vertebrate cilia was verified by examination of zebrafish flanders mutants. ccdc151ts272a (flanders) was generated in the Tübingen ENU mutagenesis screen.56 flanders mutants present morphologically with a ventral body curvature and kidney cysts (Figure 2A), characteristic of mutations that affect ciliary motility in zebrafish. We mapped the flanders mutation to a 2.4 Mb region on chromosome 6 and sequenced exons from candidate genes in this region. A c.631T\u003eA substitution was discovered in exon 6 of ccdc151 (RefSeq NM_001077369.2) that is predicted to introduce a premature stop codon at lysine 211 of the 545 amino acid protein (p.Lys211∗) (Figure 2B). Consistent with what was previously reported,54 whole mount in situ hybridization (WISH) analysis identified ccdc151 expression restricted to tissues that contain motile cilia in zebrafish including the left-right organizer (Kupffer’s vesicle [KV]), the otic vesicle, and the pronephric tubules (Figure 2C). Further support that ccdc151 is the gene mutated in flanders was provided by in situ hybridization showing evidence for nonsense-mediated decay of the transcript in embryos genotyped as mutant, which entirely lacked expression (Figure 2D). In addition, the flanders phenotype could be rescued by injection of ccdc151 RNA (Figure S2), and a phenocopy of the flanders mutant phenotype was generated by antisense morpholino injection (Figures S2 and S3).\nTo examine left-right patterning in flanders mutants and ccdc151 morphants, expression of the nodal gene southpaw (spaw) and the positioning of the visceral organs (heart, liver, and pancreas) were examined (Figure S3). Whereas wild-type siblings express spaw in the left lateral plate mesoderm and display situs solitus, flanders mutant embryos and ccdc151 morphants show randomization of spaw expression, situs inversus, and heterotaxic organ placement (Figures 2E, 2F, and S3). To explore the effect in flanders mutants and ccdc151 morphants on ciliary motility, cilia were imaged using high-speed videomicroscopy in the KV and developing kidney. In flanders mutants, cilia in the KV moved irregularly, occasionally switching direction, or were static (Movies S1 and S2). In the pronephric tubules, ciliary motility appeared less affected because cilia were able to bundle and beat regularly; however, the mutant cilia beat with significantly reduced beat frequency compared to those in unaffected sibling embryos (Figures 2G and S2; Movies S3 and S4). TEM ultrastructural analysis of pronephric cilia from flanders mutants revealed a loss of the ciliary outer dynein arms (Figure 2H).\n\nA Putative ccdc151 Ortholog Is Required for Proper Cilia Function in Planarians\nInterestingly, a putative ccdc151 ortholog (amino acid similarity 56%; identity 33%) is also required for proper cilia function in planarians (Figure S4). Planarians are flatworms that move on a ventral ciliated epithelium (Figures S4A–S4A′) and defects in cilia function cause characteristic locomotion defects.57 Hence, planarian locomotion represents a simple readout for cilia dysfunction. We found that ccdc151 RNAi-treated planarians (Figure S4C) had severely reduced locomotive ability, moving only by contracting their muscles rather than gliding (Figures S4D–S4D′; Movies S5 and S6). TEM analysis revealed a loss of ODAs in the mutant axonemes compared to control axonemes, consistent with the findings in zebrafish and mouse CCDC151 mutants (Figure S4E). Together, these data support an evolutionarily conserved role for ccdc151 in establishing proper ciliary motility in vertebrates and invertebrates.\n\nCcdc151 Is Expressed at the Mouse Embryonic Node and Ccdc151-Deficient Mice Exhibit Immotile and Dyskinetic Cilia and Laterality Defects\nTo further examine the developmental aspects of Ccdc151 function, we performed WISH on mouse embryos examined at embryonic day E7.5 when the node is present. This identified specific expression of Ccdc151 in the ventral node, consistent with the zebrafish analysis (Figure 3A). To examine the consequence of Ccdc151 deficiency on embryonic development, we further investigated the mouse model using a mutant, Snowball (Snbl), which was recovered from a large-scale mouse mutagenesis screen for mutations causing congenital heart defects.58 Whole mouse exome sequencing analysis in Snowball homozygous mutants identified five homozygous coding variants (Table S5) that were genotyped across all the mutants from the same family. However, a single-base substitution in the highly conserved +2 canonical splice donor site of Ccdc151 (RefSeq NM_029939.3) exon 6, c.828+2A\u003eG (Figure 3B), was the only candidate mutation that was homozygous in all the mutants, thus indicating it is disease causing (Table S5). This substitution causes anomalous splicing, as shown by RT-PCR using tracheal RNA isolated from wild-type and homozygous Snowball mutants and primers spanning the gene, which all yielded no RT-PCR products in Ccdc151Snbl mutants compared to controls (Figure S5). The Ccdc151Snbl allele therefore appears to convey a loss-of-function mutation subject to nonsense-mediated decay similar to that of zebrafish flanders mutants. Analysis of the tracheal airway epithelia by high-speed videomicroscopy showed largely immotile cilia in Ccdc151Snbl mutants as compared to the normal rapid synchronous beating of the wild-type littermates (Movie S7). Similarly, the ependymal cilia lining the brain ventricles of mutants were largely immotile, with occasional patches exhibiting very slow and stiff ciliary motion, while rapid synchronous ciliary motion was observed in the ependymal tissue of wild-type littermates (Movie S8). TEM of tracheal cilia from homozygous mutant Ccdc151Snbl mice showed a specific loss of the ciliary outer dynein arms (Figure 3C). We also performed high-resolution IF microscopy of Snowball tracheal epithelia using antibodies to mouse axonemal dynein heavy chain DNAH5, which is a subunit of the ODA complexes, present in both the distal and proximal ODA types present in respiratory cilia.59 This showed that DNAH5 is undetectable in Ccdc151Snbl cilia, consistent with a defect of ODA assembly in the ciliary axonemes (Figure 3D).\nPhenotyping analysis of homozygous Ccdc151Snbl mutant animals showed a spectrum of features with three distinct laterality phenotypes, as detailed in Table S6. Mutants displayed either situs solitus with normal visceral organ situs (Figure 3E, panel I), situs inversus totalis with mirror-image symmetric organ situs (Figure 3E, panel II), or heterotaxy with discordant or randomized organ situs (Figure 3E, panel III). In the latter case of more complex heterotaxy, a typical mutant exhibited normal heart orientation (levocardia) and lung lobation, but inverted liver lobation with dextrogastria (Figure 3E, panel III). Among mutants surviving to term, 33% exhibited heterotaxy and 66% had either situs inversus or situs solitus. These findings are consistent with observations from other mouse models of PCD such as Dnah5, Armc4, and Dyx1c1 mutants.21,27,48 Consistent with other PCD mouse models, congenital heart defects observed in Ccdc151Snbl mutants included dextrocardia with a duplicated inferior vena cava (Figure 3E, panel V). This was confirmed by histopathology examination of intracardiac anatomy with 3D reconstruction using episcopic confocal microscopy, which also revealed a muscular ventricular septal defect (VSD) akin to that seen in the affected individual OP-675 carrying CCDC151 mutations (Figure 3E, panel VIII). In another heterotaxic mutant, a coronary fistula was detected by videomicroscopy of the contracting heart (Figure 3E, panel VI and Movie S9) and confirmed by episcopic confocal microscopy 3D reconstruction (Figure 3E, panel IX). As documented in detail in Table S6, in addition to abdominal inversion and abnormal lung lobation and bronchial branching, other heart defects were noted including inverted outflow tract. Together, these findings confirm Snowball to be an informative PCD mouse model.\n\nCCDC151 Localizes to Respiratory Ciliary Axonemes and CCDC151 Mutations Are Associated with Loss of the Ciliary Outer Dynein Arms and Ciliary Immotility in Humans\nTo further explore the role of CCDC151 in human disease, we next examined protein localization in respiratory ciliated cells. We first screened protein lysates isolated from human nasal respiratory epithelial cells using commercially available rabbit polyclonal antibodies directed against CCDC151. Immunoblot analysis showed that the antibodies specifically recognize CCDC151, detecting a single protein band of the predicted molecular weight (∼64 kDa) (Figure 4A). We then used this antibody to analyze the subcellular localization of the protein in human motile respiratory cilia. IF showed that CCDC151 localizes to the axonemes of wild-type human respiratory epithelial cells, overlapping with an acetylated α-tubulin marker of ciliary axonemes. However, the protein was undetectable in the respiratory cilia of individuals OP-675 and OP-1255, consistent with the predicted loss-of-function consequences of the CCDC151 nonsense mutations they carry (Figure 4B). We used high-speed videomicroscopy to analyze respiratory ciliary beating in individuals with CCDC151 mutations. Both individuals OP-675 and OP-1255 had completely immotile cilia compared to the coordinated synchronous beating of cilia from unaffected control individuals, recapitulating the functional defects of the Ccdc151Snbl mice (Movies S10, S11, and S12).\nUltrastructural analysis by TEM of respiratory ciliary axonemes from individuals carrying CCDC151 mutations showed a loss of the outer dynein arms (mean of ODAs detected: 0.8–0.9) from ciliary axonemes compared to those of unaffected control individuals (ODA mean: 7.5–9) (Figure 5C). These results are consistent with the ultrastructural ciliary phenotype of ccdc151ts272a (flanders) mutants, ccdc151 RNAi planarians, and Ccdc151Snbl mice (Figures 2H, S4E, and 3C). We further examined this defect at the molecular level by immunofluorescence staining of the respiratory cells of individuals OP-675 and OP-1255 using antibodies directed against two established markers of human dynein arm integrity, the ODA marker DNAH5 and IDA marker DNALI1 (which is a light intermediate dynein associated with some IDAs). DNAH5 was undetectable in the axonemes of CCDC151 mutant individuals, suggesting that CCDC151 deficiency likely causes a disruption of axonemal ODA assembly (Figure 5A). In contrast, DNALI1 correctly localized to the axonemes of both individuals’ respiratory cells. This marker showed a similar distribution to that of control individual’s cilia (Figure 5B), suggesting that CCDC151 mutations do not alter assembly of IDA proteins.\nTogether, the TEM and IF data indicate that the axonemes of CCDC151-deficient cilia have ODA defects but that DNALI1-related IDA assembly is undisturbed. We also examined the integrity of the ciliary nexin dynein regulatory complexes (N-DRC) in CCDC151 mutant cilia by immunolocalization using antibodies directed against an integral N-DRC component, GAS8 (human DRC4).33–35 Similarly to DNALI1, GAS8 correctly localized to mutant ciliary axonemes, indicating that N-DRC assembly is not affected (Figure 5B).\n\nCCDC151 Plays a Role in Assembly of the Outer Dynein Arm Docking Complex in Addition to Its Role in Outer Dynein Arm Assembly\nTo further understand the functional role of CCDC151 in ODA assembly, we also studied the localization of CCDC114, which is an ODA-DC subunit responsible for axonemal microtubule attachment of the ODAs16 in CCDC151 mutant cilia. We found that CCDC114 was undetectable in the respiratory cilia of CCDC151-mutant individuals compared to the normal axonemal localization of CCDC114 in respiratory cilia from unaffected controls (Figure 6A). This suggests that the axonemal localization of CCDC114 is CCDC151 dependent. Since the localization of the ODA-DC-related ARMC4 is known to be CCDC114 dependent,21 we also studied ARMC4 localization in CCDC151 mutant cilia. ARMC4 was also undetectable in the respiratory cilia of CCDC151 mutant individuals, indicating that similarly to CCDC114, the axonemal localization of ARMC4 is CCDC151 dependent (Figure 6B).\nConsidering the similarities in phenotype caused by CCDC114 and CCDC151 mutations with regard to ODA defects,16 we tested for possible interactions between these proteins. Using myc- and FLAG-tagged proteins that were coexpressed in HEK293 cells, we found by coimmunoprecipitation that CCDC151 interacted with CCDC114 (Figure 6C), but not DNAI1, DNAI2, and DNAL1, whose mutations also cause ODA defects (data not shown). We confirmed the reciprocal interaction between CCDC114 and CCDC151 by yeast two-hybrid analysis 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