Bi-allelic mutations of CAMSAP1 result in teratozoospermia with sperm head and flagella defects in humans

STUDY QUESTION: What is the role of calmodulin-regulated spectrin-associated protein 1 (CAMSAP1) in human spermatogenesis? SUMMARY ANSWER: Biallelic CAMSAP1 variants cause human male infertility with multiple sperm head deformities and MMAF-like phenotypes by disrupting manchette microtubule dynamics and the microtubule-spectrin-actin scaffold essential for sperm head shaping. WHAT IS KNOWN ALREADY: Sperm head shaping requires coordinated acrosome formation, chromatin condensation, and manchette-driven nuclear remodeling. Although several genes have been implicated in globozoospermia, macrozoospermia, and multiple morphological abnormalities of the sperm flagella (MMAF), the genetic causes of other forms of abnormal sperm head morphology remain largely unknown. Camsap1-deficient mice exhibit severe spermatogenic defects, including abnormal sperm head shaping and flagellar malformations resulting from disruption of the acrosome-acroplaxome-manchette complex, ultimately leading to male infertility. However, whether CAMSAP1 plays a conserved role in human sperm development has not been established. STUDY DESIGN, SIZE, DURATION: The CAMSAP1 variants were identified by whole-exome sequencing in a cohort of 3757 male infertility patients, and all participants were recruited from 2015 to 2025. In vitro functional assays, immunoprecipitation-mass spectrometry, and sperm morphological analyses were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three infertile men with severely impaired sperm morphology and motility were recruited. Bioinformatic assessment, cell-based expression assays, immunostaining, and co-immunoprecipitation (Co-IP) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using human testicular extracts were used to characterize the functional impact of the variants. MAIN RESULTS AND THE ROLE OF CHANCE: Homozygous CAMSAP1 variants were identified in two individuals from consanguineous families (Chinese and Pakistani), and compound heterozygous missense variants in a third individual from a non-consanguineous Chinese family through whole-exome sequencing and further confirmed by Sanger sequencing. Sperm from affected individuals displayed MMAF phenotypes and diverse head abnormalities, including pyriform, amorphous, small, and globozoospermic forms. All four CAMSAP1 variants were predicted to be damaging and exhibited markedly reduced protein stability in vitro. Proteomic analysis revealed that CAMSAP1 interacts with proteins involved in actin cytoskeleton organization, acrosomal vesicle trafficking, microtubule bundle assembly, and calmodulin regulation. Mechanistically, CAMSAP1 associates with CALM1 and SPTBN1 to couple manchette microtubules with the perinuclear spectrin-actin network, ensuring proper force transmission for nuclear shaping. Mutations disrupted these interactions, leading to defective manchette architecture, abnormal acrosome assembly, and nuclear deformation. LIMITATIONS, REASONS FOR CAUTION: Although CRISPR-Cas9 knock-in cell lines carrying patient-derived CAMSAP1 variants were generated, this in vitro model could not reproduce the highly dynamic processes of manchette assembly and nuclear remodeling that occur uniquely during spermiogenesis, and therefore cannot fully capture the mechanistic sequence leading to sperm head deformation. Additionally, the number of affected individuals remains limited, and larger multi-center, multi-ethnic cohorts will be required to validate the pathogenicity of CAMSAP1 variants and further delineate the associated clinical spectrum. WIDER IMPLICATIONS OF THE FINDINGS: This work identifies CAMSAP1 as a previously unrecognized cause of human asthenoteratozoospermia and expands the genetic landscape of sperm head deformities. The findings underscore the essential role of CAMSAP1 in manchette-mediated nuclear remodeling and provide new insights for genetic diagnosis, counseling, and management of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (grant no. 82301815), the National Key R&D Program of China (grant no. 2022YFC2702603), the Sichuan Province Science and Technology Innovation Talent Project (grant no. 2024JDRC0006), the 2024 Key Open Project of Sichuan Provincial Key Laboratory for Human Disease Gene Research (grant no. 2024kflx002), and the China Postdoctoral Science Foundation (grant nos. 2023M732468 and GZC20231835). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.