Lack of CFAP54 causes primary ciliary dyskinesia in a mouse model and human patients

Abstract

Primary ciliary dyskinesia (PCD) is a highly heterogeneous recessive inherited disorder. FAP54, the homolog of CFAP54 in Chlamydomonas reinhardtii, was previously demonstrated as the C1d projection of the central microtubule apparatus of flagella. A Cfap54 knockout mouse model was then reported to have PCD-relevant phenotypes. Through whole-exome sequencing, compound heterozygous variants c.2649_2657delinC (p. E883Dfs*47) and c.7312_7313insCGCAGGCTGAATTCTTGG (p. T2438delinsTQAEFLA) in a new suspected PCD-relevant gene, CFAP54, were identified in an individual with PCD. Two missense variants, c.4112A>C (p. E1371A) and c.6559C>T (p. P2187S), in CFAP54 were detected in another unrelated patient. In this study, a minigene assay was conducted on the frameshift mutation showing a reduction in mRNA expression. In addition, a CFAP54 in-frame variant knock-in mouse model was established, which recapitulated the typical symptoms of PCD, including hydrocephalus, infertility, and mucus accumulation in nasal sinuses. Correspondingly, two missense variants were deleterious, with a dramatic reduction in mRNA abundance from bronchial tissue and sperm. The identification of PCD-causing variants of CFAP54 in two unrelated patients with PCD for the first time provides strong supportive evidence that CFAP54 is a new PCD-causing gene. This study further helps expand the disease-associated gene spectrum and improve genetic testing for PCD diagnosis in the future.

Keywords

primary ciliary dyskinesia / CFAP54 / cilia

References

[1] Mirra V, Werner C, Santamaria F. Primary ciliary dyskinesia: an update on clinical aspects, genetics, diagnosis, and future treatment strategies. Front Pediatr 2017; 5: 135

[2] ZariwalaMAKnowles MRLeighMW. Primary Ciliary Dyskinesia. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Stephens K, Amemiya A. GeneReviews. Seattle (WA): University of Washington, 1993

[3] Kurkowiak M, Ziętkiewicz E, Witt M. Recent advances in primary ciliary dyskinesia genetics. J Med Genet 2015; 52(1): 1–9

[4] Yoshiba S, Shiratori H, Kuo IY, Kawasumi A, Shinohara K, Nonaka S, Asai Y, Sasaki G, Belo JA, Sasaki H, Nakai J, Dworniczak B, Ehrlich BE, Pennekamp P, Hamada H. Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2. Science 2012; 338(6104): 226–231

[5] Knowles MR, Zariwala M, Leigh M. Primary ciliary dyskinesia. Clin Chest Med 2016; 37(3): 449–461

[6] Lucas JS, Davis SD, Omran H, Shoemark A. Primary ciliary dyskinesia in the genomics age. Lancet Respir Med 2020; 8(2): 202–216

[7] Horani A, Ferkol TW. Understanding primary ciliary dyskinesia and other ciliopathies. J Pediatr 2021; 230: 15–22.e1

[8] Brown JM, Dipetrillo CG, Smith EF, Witman GBA. A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus. J Cell Sci 2012; 125(Pt 16): 3904–3913

[9] Takeuchi K, Kitano M, Ishinaga H, Kobayashi M, Ogawa S, Nakatani K, Masuda S, Nagao M, Fujisawa T. Recent advances in primary ciliary dyskinesia. Auris Nasus Larynx 2016; 43(3): 229–236

[10] Mitchell DR, Smith B. Analysis of the central pair microtubule complex in Chlamydomonas reinhardtii. Methods Cell Biol 2009; 92: 197–213

[11] Pazour GJ, Agrin N, Leszyk J, Witman GB. Proteomic analysis of a eukaryotic cilium. J Cell Biol 2005; 170(1): 103–113

[12] DiPetrillo CG, Smith EF. Pcdp1 is a central apparatus protein that binds Ca²⁺-calmodulin and regulates ciliary motility. J Cell Biol 2010; 189(3): 601–612

[13] McKenzie CW, Craige B, Kroeger TV, Finn R, Wyatt TA, Sisson JH, Pavlik JA, Strittmatter L, Hendricks GM, Witman GB, Lee L. CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice. Mol Biol Cell 2015; 26(18): 3140–3149

[14] Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009; 25(14): 1754–1760

[15] Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009; 25(16): 2078–2079

[16] 1000 Genomes Project Consortium; Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, Korbel JO, Marchini JL, McCarthy S, McVean GA, Abecasis GR. A global reference for human genetic variation. Nature 2015; 526(7571): 68–74

[17] Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, Collins RL, Laricchia KM, Ganna A, Birnbaum DP, Gauthier LD, Brand H, Solomonson M, Watts NA, Rhodes D, Singer-Berk M, England EM, Seaby EG, Kosmicki JA, Walters RK, Tashman K, Farjoun Y, Banks E, Poterba T, Wang A, Seed C, Whiffin N, Chong JX, Samocha KE, Pierce-Hoffman E, Zappala Z, O'Donnell-Luria AH, Minikel EV, Weisburd B, Lek M, Ware JS, Vittal C, Armean IM, Bergelson L, Cibulskis K, Connolly KM, Covarrubias M, Donnelly S, Ferriera S, Gabriel S, Gentry J, Gupta N, Jeandet T, Kaplan D, Llanwarne C, Munshi R, Novod S, Petrillo N, Roazen D, Ruano-Rubio V, Saltzman A, Schleicher M, Soto J, Tibbetts K, Tolonen C, Wade G, Talkowski ME; Genome Aggregation Database Consortium; Neale BM, Daly MJ, MacArthur DG. The mutational constraint spectrum quantified from variation in 141 456 humans. Nature 2020; 581(7809): 434–443

[18] Fu W, O'Connor TD, Jun G, Kang HM, Abecasis G, Leal SM, Gabriel S, Rieder MJ, Altshuler D, Shendure J, Nickerson DA, Bamshad MJ; NHLBI Exome Sequencing Project; Akey JM. Analysis of 6515 exomes reveals the recent origin of most human protein-coding variants. Nature 2013; 493(7431): 216–220

[19] Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 2010; 38(16): e164

[20] Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009; 4(7): 1073–1081

[21] Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. A method and server for predicting damaging missense mutations. Nat Methods 2010; 7(4): 248–249

[22] Schwarz JM, Rödelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010; 7(8): 575–576

[23] Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 2014; 46(3): 310–315

[24] Amberger JS, Hamosh A. Searching Online Mendelian Inheritance in Man (OMIM): a knowledgebase of human genes and genetic phenotypes. Curr Protoc Bioinformatics 2017; 58: 1.2.1–1.2.12

[25] Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000; 28(1): 27–30

[26] Landrum MJ, Lee JM, Benson M, Brown G, Chao C, Chitipiralla S, Gu B, Hart J, Hoffman D, Hoover J, Jang W, Katz K, Ovetsky M, Riley G, Sethi A, Tully R, Villamarin-Salomon R, Rubinstein W, Maglott DR. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res 2016; 44(D1): D862–D868

[27] Schaefer CF, Anthony K, Krupa S, Buchoff J, Day M, Hannay T, Buetow KH. PID: the pathway interaction database. Nucleic Acids Res 2009; 37(Database issue suppl_1): D674–D679

[28] Stenson PD, Mort M, Ball EV, Evans K, Hayden M, Heywood S, Hussain M, Phillips AD, Cooper DN. The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum Genet 2017; 136(6): 665–677

[29] Fabregat A, Jupe S, Matthews L, Sidiropoulos K, Gillespie M, Garapati P, Haw R, Jassal B, Korninger F, May B, Milacic M, Roca CD, Rothfels K, Sevilla C, Shamovsky V, Shorser S, Varusai T, Viteri G, Weiser J, Wu G, Stein L, Hermjakob H, D’Eustachio P. The Reactome Pathway Knowledgebase. Nucleic Acids Res 2018; 46(D1): D649–D655

[30] Kim W, Han TH, Kim HJ, Park MY, Kim KS, Park RW. An automated measurement of ciliary beating frequency using a combined optical flow and peak detection. Healthc Inform Res 2011; 17(2): 111–119

[31] Smith CM, Djakow J, Free RC, Djakow P, Lonnen R, Williams G, Pohunek P, Hirst RA, Easton AJ, Andrew PW, O’Callaghan C. ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software. Cilia 2012; 1(1): 14

[32] Sampaio P, da Silva MF, Vale I, Roxo-Rosa M, Pinto A, Constant C, Pereira L, Quintão CM, Lopes SS. CiliarMove: new software for evaluating ciliary beat frequency helps find novel mutations by a Portuguese multidisciplinary team on primary ciliary dyskinesia. ERJ Open Res 2021; 7(1): 00792–2020

[33] Yi WJ, Park KS, Min YG, Sung MW. Distribution mapping of ciliary beat frequencies of respiratory epithelium cells using image processing. Med Biol Eng Comput 1997; 35(6): 595–599

[34] Sisson JH, Stoner JA, Ammons BA, Wyatt TA. All-digital image capture and whole-field analysis of ciliary beat frequency. J Microsc 2003; 211(2): 103–111

[35] Shapiro AJ, Davis SD, Polineni D, Manion M, Rosenfeld M, Dell SD, Chilvers MA, Ferkol TW, Zariwala MA, Sagel SD, Josephson M, Morgan L, Yilmaz O, Olivier KN, Milla C, Pittman JE, Daniels MLA, Jones MH, Janahi IA, Ware SM, Daniel SJ, Cooper ML, Nogee LM, Anton B, Eastvold T, Ehrne L, Guadagno E, Knowles MR, Leigh MW, Lavergne V; American Thoracic Society Assembly on Pediatrics. Diagnosis of primary ciliary dyskinesia. An official American Thoracic Society clinical practice guideline. Am J Respir Crit Care Med 2018; 197(12): e24–e39

[36] Teves ME, Nagarkatti-Gude DR, Zhang Z, Strauss JF 3rd. Mammalian axoneme central pair complex proteins: Broader roles revealed by gene knockout phenotypes. Cytoskeleton (Hoboken) 2016; 73(1): 3–22

[37] Collins SA, Gove K, Walker W, Lucas JSA. Nasal nitric oxide screening for primary ciliary dyskinesia: systematic review and meta-analysis. Eur Respir J 2014; 44(6): 1589–1599

[38] Kott E, Legendre M, Copin B, Papon JF, Dastot-Le Moal F, Montantin G, Duquesnoy P, Piterboth W, Amram D, Bassinet L, Beucher J, Beydon N, Deneuville E, Houdouin V, Journel H, Just J, Nathan N, Tamalet A, Collot N, Jeanson L, Le Gouez M, Vallette B, Vojtek AM, Epaud R, Coste A, Clement A, Housset B, Louis B, Escudier E, Amselem S. Loss-of-function mutations in RSPH1 cause primary ciliary dyskinesia with central-complex and radial-spoke defects. Am J Hum Genet 2013; 93(3): 561–570

[39] Knowles MR, Ostrowski LE, Leigh MW, Sears PR, Davis SD, Wolf WE, Hazucha MJ, Carson JL, Olivier KN, Sagel SD, Rosenfeld M, Ferkol TW, Dell SD, Milla CE, Randell SH, Yin W, Sannuti A, Metjian HM, Noone PG, Noone PJ, Olson CA, Patrone MV, Dang H, Lee HS, Hurd TW, Gee HY, Otto EA, Halbritter J, Kohl S, Kircher M, Krischer J, Bamshad MJ, Nickerson DA, Hildebrandt F, Shendure J, Zariwala MA. Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype. Am J Respir Crit Care Med 2014; 189(6): 707–717

[40] Ibañez-Tallon I, Gorokhova S, Heintz N. Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus. Hum Mol Genet 2002; 11(6): 715–721

[41] Becker-Heck A, Zohn IE, Okabe N, Pollock A, Lenhart KB, Sullivan-Brown J, McSheene J, Loges NT, Olbrich H, Haeffner K, Fliegauf M, Horvath J, Reinhardt R, Nielsen KG, Marthin JK, Baktai G, Anderson KV, Geisler R, Niswander L, Omran H, Burdine RD. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation. Nat Genet 2011; 43(1): 79–84

[42] Zou W, Lv Y, Liu ZI, Xia P, Li H, Jiao J. Loss of Rsph9 causes neonatal hydrocephalus with abnormal development of motile cilia in mice. Sci Rep 2020; 10(1): 12435

[43] Kempeneers C, Seaton C, Garcia Espinosa B, Chilvers MA. Ciliary functional analysis: beating a path towards standardization. Pediatr Pulmonol 2019; 54(10): 1627–1638

[44] Bustamante-Marin XM, Yin WN, Sears PR, Werner ME, Brotslaw EJ, Mitchell BJ, Jania CM, Zeman KL, Rogers TD, Herring LE, Refabért L, Thomas L, Amselem S, Escudier E, Legendre M, Grubb BR, Knowles MR, Zariwala MA, Ostrowski LE. Lack of GAS2L2 causes PCD by impairing cilia orientation and mucociliary clearance. Am J Hum Genet 2019; 104(2): 229–245

[45] ParrillaEArmengot MMataMCortijoJRieraJ HuesoJLMoratal D. Optical flow method in phase-contrast microscopy images for the diagnosis of primary ciliary dyskinesia through measurement of ciliary beat frequency. Preliminary results. 2012 9th IEEE International Symposium on Biomedical Imaging (ISBI), Barcelona, Spain, 2012, 1655–1658 doi:10.1109/ISBI.2012.6235895

[46] Petriman NA, Lorentzen E. Structural insights into the architecture and assembly of eukaryotic flagella. Microb Cell 2020; 7(11): 289–299

[47] Ishikawa T. 3D structure of eukaryotic flagella/cilia by cryo-electron tomography. Biophysics (Nagoya-Shi) 2013; 9(0): 141–148

[48] Olbrich H, Cremers C, Loges NT, Werner C, Nielsen KG, Marthin JK, Philipsen M, Wallmeier J, Pennekamp P, Menchen T, Edelbusch C, Dougherty GW, Schwartz O, Thiele H, Altmüller J, Rommelmann F, Omran H. Loss-of-function GAS8 mutations cause primary ciliary dyskinesia and disrupt the nexin-dynein regulatory complex. Am J Hum Genet 2015; 97(4): 546–554

[49] Wirschell M, Olbrich H, Werner C, Tritschler D, Bower R, Sale WS, Loges NT, Pennekamp P, Lindberg S, Stenram U, Carlén B, Horak E, Köhler G, Nürnberg P, Nürnberg G, Porter ME, Omran H. The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans. Nat Genet 2013; 45(3): 262–268

[50] Horani A, Brody SL, Ferkol TW, Shoseyov D, Wasserman MG, Ta-shma A, Wilson KS, Bayly PV, Amirav I, Cohen-Cymberknoh M, Dutcher SK, Elpeleg O, Kerem E. CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia. PLoS One 2013; 8(8): e72299

[51] Lucas JS, Barbato A, Collins SA, Goutaki M, Behan L, Caudri D, Dell S, Eber E, Escudier E, Hirst RA, Hogg C, Jorissen M, Latzin P, Legendre M, Leigh MW, Midulla F, Nielsen KG, Omran H, Papon JF, Pohunek P, Redfern B, Rigau D, Rindlisbacher B, Santamaria F, Shoemark A, Snijders D, Tonia T, Titieni A, Walker WT, Werner C, Bush A, Kuehni CE. European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia. Eur Respir J 2017; 49(1): 1601090

[52] Horani A, Ferkol TW, Dutcher SK, Brody SL. Genetics and biology of primary ciliary dyskinesia. Paediatr Respir Rev 2016; 18: 18–24

[53] Han L, Rao Q, Yang R, Wang Y, Chai P, Xiong Y, Zhang K. Cryo-EM structure of an active central apparatus. Nat Struct Mol Biol 2022; 29(5): 472–482

[54] Gui M, Wang X, Dutcher SK, Brown A, Zhang R. Ciliary central apparatus structure reveals mechanisms of microtubule patterning. Nat Struct Mol Biol 2022; 29(5): 483–492

[55] DiPetrillo CG, Smith EF. The Pcdp1 complex coordinates the activity of dynein isoforms to produce wild-type ciliary motility. Mol Biol Cell 2011; 22(23): 4527–4538

[56] Lee L, Campagna DR, Pinkus JL, Mulhern H, Wyatt TA, Sisson JH, Pavlik JA, Pinkus GS, Fleming MD. Primary ciliary dyskinesia in mice lacking the novel ciliary protein Pcdp1. Mol Cell Biol 2008; 28(3): 949–957

[57] Bustamante-Marin XM, Shapiro A, Sears PR, Charng WL, Conrad DF, Leigh MW, Knowles MR, Ostrowski LE, Zariwala MA. Identification of genetic variants in CFAP221 as a cause of primary ciliary dyskinesia. J Hum Genet 2020; 65(2): 175–180

[58] Liu C, Tu C, Wang L, Wu H, Houston BJ, Mastrorosa FK, Zhang W, Shen Y, Wang J, Tian S, Meng L, Cong J, Yang S, Jiang Y, Tang S, Zeng Y, Lv M, Lin G, Li J, Saiyin H, He X, Jin L, Touré A, Ray PF, Veltman JA, Shi Q, O’Bryan MK, Cao Y, Tan YQ, Zhang F. Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility. Am J Hum Genet 2021; 108(2): 309–323

[59] Sha Y, Wei X, Ding L, Ji Z, Mei L, Huang X, Su Z, Wang W, Zhang X, Lin S. Biallelic mutations of CFAP74 may cause human primary ciliary dyskinesia and MMAF phenotype. J Hum Genet 2020; 65(11): 961–969

[60] Thomas L, Bouhouche K, Whitfield M, Thouvenin G, Coste A, Louis B, Szymanski C, Bequignon E, Papon JF, Castelli M, Lemullois M, Dhalluin X, Drouin-Garraud V, Montantin G, Tissier S, Duquesnoy P, Copin B, Dastot F, Couvet S, Barbotin AL, Faucon C, Honore I, Maitre B, Beydon N, Tamalet A, Rives N, Koll F, Escudier E, Tassin AM, Touré A, Mitchell V, Amselem S, Legendre M. TTC12 loss-of-function mutations cause primary ciliary dyskinesia and unveil distinct dynein assembly mechanisms in motile cilia versus flagella. Am J Hum Genet 2020; 106(2): 153–169

[61] Sironen A, Kotaja N, Mulhern H, Wyatt TA, Sisson JH, Pavlik JA, Miiluniemi M, Fleming MD, Lee L. Loss of SPEF2 function in mice results in spermatogenesis defects and primary ciliary dyskinesia. Biol Reprod 2011; 85(4): 690–701

Acknowledgements

The authors thank all the patients and other participants in this study. This study was supported by the National Key Research and Development Program of China (No. 2016YFC0901502 to Kai-Feng Xu; No. 2016YFC0905100 to Xue Zhang; No. 2017YFC1001201 to Yaping Liu), the National Natural Science Foundation of China (NSFC) (No. 81788101 to Xue Zhang; No. 31271345 to Yaping Liu), and the CAMS Initiative for Medical Sciences (CIFMS) (Nos. 2021-1-I2M-018 and 2016-I2M-1-002 to Xue Zhang and Yaping Liu; Nos. 2020-I2M-C&T-B-002 and 2018-I2M-1-003 to Xinlun Tian).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11684-023-0997-7 and is accessible for authorized users.

Compliance with ethics guidelines

Conflicts of interest Xinyue Zhao, Haijun Ge, Wenshuai Xu, Chongsheng Cheng, Wangji Zhou, Yan Xu, Junping Fan, Yaping Liu, and Xinlun Tian declare that they have no conflict of interest. Kai-Feng Xu and Xue Zhang are members of the Editorial Board of Frontiers of Medicine, who were excluded from the peer-review process and all editorial decisions related to the acceptance and publication of this article. Peer-review was handled independently by the other editors to minimise bias.

The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards, and it was approved by the Institutional Review Board committee at PUMCH. Informed consent was obtained from all patients for being included in the study. All institutional and national guidelines for the care and use of laboratory animals were followed.

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