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Novel homozygous CFAP69 mutations in humans and mice cause severe asthenoteratospermia with multiple morphological abnormalities of the sperm flagella
  1. Xiaojin He1,2,3,
  2. Weiyu Li4,5,6,
  3. Huan Wu1,2,3,
  4. Mingrong Lv1,2,3,
  5. Wangjie Liu4,6,
  6. Chunyu Liu4,6,
  7. Fuxi Zhu1,2,3,
  8. Caihua Li7,
  9. Youyan Fang1,2,
  10. Chenyu Yang8,
  11. Huiru Cheng2,
  12. Junqiang Zhang2,
  13. Jing Tan1,2,3,
  14. Tingting Chen9,
  15. Dongdong Tang1,2,3,
  16. Bing Song1,2,3,
  17. Xue Wang1,2,3,
  18. Xiaomin Zha1,2,3,
  19. Hongyan Wang4,6,
  20. Zhaolian Wei1,2,3,
  21. Shenmin Yang10,
  22. Hexige Saiyin4,
  23. Ping Zhou1,2,3,
  24. Li Jin4,
  25. Jian Wang11,
  26. Zhiguo Zhang1,2,3,
  27. Feng Zhang4,5,6,
  28. Yunxia Cao1,2,3
  1. 1 Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China
  2. 2 Anhui Province Key Laboratory of Reproductive Health and Genetics, Anhui Medical University, Hefei, China
  3. 3 Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, China
  4. 4 Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering at School of Life Sciences, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai, China
  5. 5 State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China
  6. 6 NHC Key Laboratory of Reproduction Regulation, Shanghai Institute of Planned Parenthood Research, Fudan University, Shanghai, China
  7. 7 Genesky Biotechnologies Inc, Shanghai, China
  8. 8 Center of Cryo-Electron Microscopy, Zhejiang University, Hangzhou, China
  9. 9 Department of Pathology, Anhui Medical University, Hefei, China
  10. 10 Center for Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
  11. 11 Department of Medical Genetics and Molecular Diagnostic Laboratory, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai, China
  1. Correspondence to Professor Feng Zhang, Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering at School of Life Sciences, Fudan University, Shanghai 200011, China; zhangfeng{at} and Dr Yunxia Cao, Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China; caoyunxia6{at}


Background Male infertility is a major issue of human reproduction health. Asthenoteratospermia can impair sperm motility and cause male infertility. Asthenoteratospermia with multiple morphological abnormalities of the flagella (MMAF) presents abnormal spermatozoa with absent, bent, coiled, short and/or irregular-calibre flagella. Previous studies on MMAF reported that genetic defects in cilia-related genes (eg, AKAP4, DNAH1, CFAP43, CFAP44 and CFAP69) are the major cause of MMAF. However, the known MMAF-associated genes are only responsible for approximately 30% to 50% of human cases. We further investigated the cases with MMAF in search of additional genes mutated in this condition.

Methods and results We conducted whole exome sequencing in a male individual with MMAF from a consanguineous Han Chinese family. Sanger sequencing was also conducted in additional individuals with MMAF. Intriguingly, a homozygous frameshift mutation (p.Leu357Hisfs*11) was identified in the gene encoding CFAP69 (cilia and flagella-associated protein 69), which is highly expressed in testis. The subsequent Sanger sequencing of the CFAP69 coding regions among 34 additional individuals with MMAF revealed a case with homozygous nonsense mutation (p.Trp216*) of CFAP69. Both of these CFAP69 loss-of-function mutations were not present in the human population genome data archived in the 1000 Genomes Project and ExAC databases, nor in 875 individuals of two Han Chinese control populations. Furthermore, we generated the knockout model in mouse orthologue Cfap69 using the CRISPR-Cas9 technology. Remarkably, male Cfap69-knockout mice manifested with MMAF phenotypes.

Conclusion Our experimental findings elucidate that homozygous loss-of-function mutations in CFAP69 can lead to asthenoteratospermia with MMAF in humans and mice.

  • CFAP69
  • flagella
  • knockout mice
  • sequencing
  • male infertility

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  • XH, WL and HW contributed equally.

  • Contributors XH: study design, molecular experiment and original draft preparation; WLi: data analysis, molecular experiment and original draft preparation; HWu: data collection, mouse knockout and original draft preparation; ML: molecular experiment; WLiu: molecular experiment and data analysis; CLiu: molecular experiment; FuZ: data analysis; CLi: data analysis; YF: molecular experiment; CY: data analysis; HC: mouse knockout and molecular experiment; JZ: mouse knockout and molecular experiment; JT: sperm analysis; TC: molecular experiment; DT: data collection; BS: data collection; XW: data collection; XZ: data collection and sperm analysis; HWa: data analysis; ZW: data analysis; SY: data analysis; HS: molecular experiment and data analysis; PZ: data analysis; LJ: data analysis; JW: data analysis; ZZ: study conception and design; FeZ: study design, data analysis and editing of the manuscript; YC: study conception and design.

  • Funding This study was supported by Foundation of the Department of Science and Technology of Anhui Province (2017070802D150), National Key Research and Development Program of China (2016YFC0905100 and 2017YFC1001100), National Natural Science Foundation of China (31625015, 11627803, 81601340, 31521003, 31571297 and 31771396), Shanghai Medical Center of Key Programs for Female Reproductive Diseases (2017ZZ01016), Foundation of the Education Department of Anhui Province (KJ2016A370), Maternal and Child Health Research Project of Jiangsu Province (F201521) and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval Ethics committee of Anhui Medical University and the Institutional review boards of the School of Life Sciences at Fudan University.

  • Provenance and peer review Not commissioned; externally peer reviewed.