Paternal USP26 mutations raise Klinefelter syndrome risk in the offspring of mice and humans

Article12 May 2021free access Source DataTransparent process Paternal USP26 mutations raise Klinefelter syndrome risk in the offspring of mice and humans Chao Liu Chao Liu orcid.org/0000-0002-8844-0697 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Hongbin Liu Hongbin Liu Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, ChinaThese authors contributed equally to this work Search for more papers by this author Haobo Zhang Haobo Zhang Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, ChinaThese authors contributed equally to this work Search for more papers by this author Lina Wang Lina Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Mengjing Li Mengjing Li Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Feifei Cai Feifei Cai Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Search for more papers by this author Xiuge Wang Xiuge Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Li Wang Li Wang Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Ruidan Zhang Ruidan Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Sijie Yang Sijie Yang Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Wenwen Liu Wenwen Liu State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yu Liang Yu Liang Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China Search for more papers by this author Liying Wang Liying Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaohui Song Xiaohui Song Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Shizhen Su Shizhen Su Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Hui Gao Hui Gao State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jing Jiang Jing Jiang Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Jinsong Li Jinsong Li Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Mengcheng Luo Mengcheng Luo Department of Tissue and Embryology, School of Basic Medical Sciences, Wuhan University, Wuhan, China Search for more papers by this author Fei Gao Fei Gao orcid.org/0000-0002-4029-6411 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qi Chen Qi Chen orcid.org/0000-0001-6353-9589 Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, CA, USA Search for more papers by this author Wei Li Corresponding Author Wei Li [email protected] orcid.org/0000-0002-6235-0749 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zi-Jiang Chen Corresponding Author Zi-Jiang Chen [email protected] orcid.org/0000-0001-6637-6631 Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China Search for more papers by this author Chao Liu Chao Liu orcid.org/0000-0002-8844-0697 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Hongbin Liu Hongbin Liu Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, ChinaThese authors contributed equally to this work Search for more papers by this author Haobo Zhang Haobo Zhang Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, ChinaThese authors contributed equally to this work Search for more papers by this author Lina Wang Lina Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Mengjing Li Mengjing Li Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Feifei Cai Feifei Cai Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Search for more papers by this author Xiuge Wang Xiuge Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Li Wang Li Wang Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Ruidan Zhang Ruidan Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Sijie Yang Sijie Yang Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Wenwen Liu Wenwen Liu State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yu Liang Yu Liang Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China Search for more papers by this author Liying Wang Liying Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaohui Song Xiaohui Song Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Shizhen Su Shizhen Su Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Search for more papers by this author Hui Gao Hui Gao State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jing Jiang Jing Jiang Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Jinsong Li Jinsong Li Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Mengcheng Luo Mengcheng Luo Department of Tissue and Embryology, School of Basic Medical Sciences, Wuhan University, Wuhan, China Search for more papers by this author Fei Gao Fei Gao orcid.org/0000-0002-4029-6411 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qi Chen Qi Chen orcid.org/0000-0001-6353-9589 Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, CA, USA Search for more papers by this author Wei Li Corresponding Author Wei Li [email protected] orcid.org/0000-0002-6235-0749 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zi-Jiang Chen Corresponding Author Zi-Jiang Chen [email protected] orcid.org/0000-0001-6637-6631 Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China Shandong Key Laboratory of Reproductive Medicine, Jinan, China Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China Search for more papers by this author Author Information Chao Liu1,2, Hongbin Liu3,4, Haobo Zhang3,4, Lina Wang1,5, Mengjing Li3,4, Feifei Cai3, Xiuge Wang1,5, Li Wang3,4, Ruidan Zhang1,5, Sijie Yang3,4, Wenwen Liu1,5, Yu Liang6, Liying Wang1,5, Xiaohui Song3,4, Shizhen Su3,4, Hui Gao1, Jing Jiang7, Jinsong Li7, Mengcheng Luo8, Fei Gao1,5, Qi Chen9, Wei Li *,1,2,5 and Zi-Jiang Chen *,3,4,6 1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Stem Cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, China 2Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China 3Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China 4Shandong Key Laboratory of Reproductive Medicine, Jinan, China 5University of Chinese Academy of Sciences, Beijing, China 6Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China 7Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China 8Department of Tissue and Embryology, School of Basic Medical Sciences, Wuhan University, Wuhan, China 9Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, CA, USA *Corresponding author. Tel: +86 10 64807529; E-mail: [email protected] *Corresponding author. Tel: +86 531 85651188; Fax: +86 531 87068226; E-mail: [email protected] The EMBO Journal (2021)40:e106864https://doi.org/10.15252/embj.2020106864 See also: L Kauppi (July 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26-mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring. Synopsis Klinefelter syndrome (KS) is believed to be not inherited, and whether there is a genetic basis for the origin of KS is unknown. Here, paternal USP26 mutations are found to produce XY spermatozoa, thus greatly increase the risk for producing KS offspring. Whole-genome sequencing-based mutational scanning in KS patients shows that USP26 variants are likely to affect the etiology of KS. Loss of Usp26 in male mice results in the production of 41, XXY offspring. The disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11, thus producing XY aneuploid spermatozoa. Paternal USP26 mutations increase the proportion of XY aneuploid sperm and cause KS offspring production. Introduction Klinefelter syndrome (KS), which is defined by the 47, XXY karyotype, is the most frequent chromosome aberration in males and affects about 1 in 500–1,000 males (Groth et al, 2013). The prevalence of KS reaches to 3–4% among infertile males and is as high as 12% in azoospermic patients (Groth et al, 2013; Bonomi et al, 2017). The major signs and symptoms of KS include small, firm testes, gynecomastia, hypergonadotropic hypogonadism, and oligospermia or azoospermia (Lanfranco et al, 2004; Groth et al, 2013; Bonomi et al, 2017). Because only very small amounts of sperm can be found in around 50% adult KS patients' testicular tissue (Groth et al, 2013; Corona et al, 2017), most KS patients are infertile under physiological conditions (Groth et al, 2013; Bonomi et al, 2017; Corona et al, 2017). Knowledge about how to prevent or treat this disorder is urgently needed. Since the 47, XXY karyotype was defined in KS patients in 1959, the pathology of KS has been found to be highly associated with X chromosome polysomy (Jacobs & Strong, 1959; Lanfranco et al, 2004; Groth et al, 2013). The presence of an extra X chromosome in KS might arise by non-disjunction during either meiosis I or meiosis II of maternal oogenesis or during meiosis I of paternal spermatogenesis (Thomas & Hassold, 2003; Lanfranco et al, 2004). Advanced maternal age is the only evidence-based risk factor for KS (Harvey et al, 1991; Tuttelmann & Gromoll, 2010), but because increased maternal age is a known strong etiological factor for other autosomal trisomies (Nicolaidis & Petersen, 1998), the maternal age-related cases of KS are likely similar to other autosomal trisomies. However, previous KS studies have shown that, in sharp contrast to most of the autosomal trisomies that largely originate from maternal meiotic defects (90%), the extra X chromosome in KS is equally likely to be of maternal or paternal origin (Hassold & Hunt, 2001; Thomas & Hassold, 2003). The fathers of KS patients also produce higher frequencies of XY sperm (Lowe et al, 2001; Eskenazi et al, 2002), highlighting that KS has a strong tendency for paternal origin among trisomy disorders. Further, although impaired recombination between paternal sex chromosomes in the pseudoautosomal region (PAR) during meiosis I has been proposed as a major cause of paternal-origin KS (Hassold et al, 1991; Thomas et al, 2000), the molecular mechanisms underlying the origin of KS are still largely unknown. Here, we conducted a genetic analysis using whole-exome sequencing (WES) in 108 unrelated KS patients and identified USP26 germline mutations that might be responsible for promoting paternal-origin KS (Fig 1). USP26 has been associated with nonobstructive azoospermia (Xia et al, 2014; Luddi et al, 2016; Ma et al, 2016; Arafat et al, 2020), and Usp26 knockout mouse models only display a very slight impact on male fertility (Felipe-Medina et al, 2019; Sakai et al, 2019; Tian et al, 2019). While we found that 2-month-old Usp26 −/ Y mice were indeed fertile, both the pregnancy rates and the litter sizes of Usp26 −/ Y mice were significantly reduced with increasing age (6-month-old mice; Fig 2A–D). Further investigation showed the knockout of Usp26 in male mice impaired sex chromosome pairing by destabilizing TEX11, which resulted in XY aneuploid spermatozoa and ultimately produced 41, XXY offspring. In fertile men, some USP26 variations increased the proportion of XY aneuploid spermatozoa, and we found that the father of two KS patients harbored a USP26-mutated haplotype. Although the deficiency of paternal Usp26 does not invariably result in the production of XXY progeny mice (or KS children for the mutated haplotype), Usp26 deletion or the mutated haplotype does greatly increase the overall frequency of KS offspring production. Figure 1. USP26 variants might be responsible for the origin of Klinefelter syndrome The karyotype of KS patients and controls. The cohorts used in this study. The first cohort that contained 108 unrelated KS patients with the classical 47, XXY karyotype was selected to perform WES, the second cohort that contained 354 unrelated KS patients was selected to perform Sanger sequencing in the coding regions of USP26, and the third cohort that contained 558 unrelated KS patients was selected to perform Sanger sequencing in the mutated haplotype (c.370–371insACA/494T>C/1423C>T) of USP26. WES data analysis pipeline. A total of 179,579 sequence variants in 20,800 genes were identified in 108 unrelated KS patients, and among them nonsense, missense, frameshift, and essential splice-site variants were further investigated. These variants were divided into four subtypes (high, likely high, medium, and low), and most of the medium to high variants were localized on the X chromosome. The genes on the X chromosome that contained more than two types of medium to high variants were selected. As the extra paternal-origin X chromosome in KS might be caused by impaired recombination between sex chromosomes during meiosis I, only the testis-specific genes ESX1, LOC101059915, and USP26 were further investigated. The variant frequencies of these three genes in the KS patients were compared with the variant frequencies of these three genes in the 1000 Genomes Project. The frequency of USP26 variants had the highest fold increase. The genomic localization of sequence variants in 108 KS patients. Genes with more than two medium- to high-level variants on the X chromosome. The testis-specific genes are labeled in red. The frequency difference between KS patients and the 1000 Genomes Project. Download figure Download PowerPoint Figure 2. Usp26-deficient mice produce 41, XXY offspring A. Sequences of the WT and Usp26 mutant alleles in mice. B. The USP26 protein was absent in Usp26 −/ Y testes. Immunoblotting of USP26 was performed in Usp26 +/ Y and Usp26 −/ Y testes. Histone 3 served as the loading control. C, D. The fertility of Usp26 −/ Y mice was reduced with increasing age. The fertility assessment experiments were performed in 2-month-old Usp26 +/ Y , Usp26 −/ Y mice (n = 5 independent experiments) and 6-month-old Usp26 +/ Y , Usp26 −/ Y mice (n = 5 independent experiments) (C). The litter sizes were observed in 2-month-old Usp26 +/ Y , Usp26 −/ Y mice (n = 14, 22 independent experiments for Usp26 +/ Y and Usp26 −/ Y , respectively) and 6-month-old Usp26 +/ Y , Usp26 −/ Y mice (n = 10, 8 independent experiments for Usp26 +/ Y and Usp26 −/ Y , respectively) (D). Red dots indicate Usp26 +/ Y mice, and green dots indicate Usp26 −/ Y mice. E. Genotyping of Usp26 −/ Y mice offspring. The upper subpanel indicates the genotyping of Usp26 −/ Y mice male offspring by using Usp26 and Sry primers. The lower subpanel indicates the genotyping of Usp26 −/ Y , Usp26 +/ Y male mice and Usp26 +/−, Usp26 +/+ female mice by using Usp26 and Sry primers. F. The wild-type (WT) and Usp26 knock out alleles in X Usp26 +X Usp26 −Y mice. G. The proportion of 41, XXY mice among the male offspring of 2-month-old Usp26 +/ Y , Usp26 −/ Y and 6-month-old Usp26 +/ Y , Usp26 −/ Y mice (n = 14, 22, 10, and 8 independent experiments for the offspring of 2-month-old Usp26 +/ Y , Usp26 −/ Y mice, 6-month-old Usp26 +/ Y , and Usp26 −/ Y mice, respectively). Red dots indicate Usp26 +/ Y mice, and green dots indicate Usp26 −/ Y mice. H, I. Two pair of X chromosomes could be detected in the 2-month-old X Usp26 +X Usp26 −Y spermatocytes. FISH analysis of Chr X-FISH (green) and Chr Y-FISH (red) was performed in WT and X Usp26 +X Usp26 −Y spermatocytes (H). Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in WT and X Usp26 +X Usp26 −Y spermatocytes (I). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. Nuclei were stained with DAPI (blue). J–L. Three sex chromosome axes could be detected in the 2-month-old X Usp26 +X Usp26 −Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) and BRCA1 (red) and SYCP3 (green) in (L) was performed in 2-month-old WT and X Usp26 +X Usp26 −Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome. Data information: In (C), (D), and (G), data are presented as mean ± SD. *P < 0.05, **P < 0.01 (Student's t-test). n.s, not significant. Numerical source data for panels (C)/(D)/(G) can be found in Table EV5. Source data are available online for this figure. Source Data for Figure 2 [embj2020106864-sup-0008-SDataFig2.zip] Download figure Download PowerPoint Results Whole-genome sequencing-based mutational scanning in KS patients To study the potential genetic basis for paternal-origin KS, we performed WES in 108 unrelated KS patients with the classical 47, XXY karyotype (1st cohort; Fig 1A and B). A total of 179,579 sequence variants were identified in the KS subset, and we subsequently investigated the nonsense, missense, frameshift, and splice-site variants among them (Fig 1C, Dataset EV1 and Table EV1). These variants were divided into four subtypes according to their frequencies, genomic localization, and potential deleterious effects (Fig 1C). Briefly, it was notable that many of the predicted deleterious variants were localized on the X chromosome (Fig 1C and D), and recalling that KS is the trisomy disorder for which paternal origin is most prevalent, and our interest was piqued by USP26 (Fig 1E and F, Table EV2), a gene for which multiple variants were detected in this cohort and which was previously reported to have a strictly testis-specific expression pattern (Wang et al, 2001). Our WES data showed six variations of USP26 in 29 of the 108 KS patients. Extending this inquiry, we sequenced the coding regions of USP26 in an additional 354 KS patients and in 272 fertile men (Table EV1 and Table EV3), which led to our identification of USP26 variants in 49 KS patients, including two synonymous mutations in 2 KS patients, three missense mutations in 12 KS patients, and a mutated haplotype in 35 KS patients. Moreover, we found that the c.191A>G[p.Y64C], c.313G>A[p.E105K], c.1030C>T[p.R344W], and c.1691A>T[p.D564V] variants only occurred in the KS patients. Thus, the USP26 variants appeared likely to impact the etiology of KS origin. XXY offspring production from Usp26-deficient mice USP26 belongs to a family of ubiquitin-specific proteases that remove ubiquitin chains from substrates (Dirac & Bernards, 2010; Lahav-Baratz et al, 2017; Ning et al, 2017), and USP26 has been reported to be associated with nonobstructive azoospermia, while others have found contradictory results, and thus, this association remains debatable (Xia et al, 2014; Zhang et al, 2015; Luddi et al, 2016; Ma et al, 2016). To determine whether USP26 mutations lead to KS offspring, we generated Usp26-knockout mice using the CRISPR-Cas9 system (Fig 2A). As Usp26 localized on the X chromosome, the Usp26 knockout male mice were called Usp26 −/ Y . We first measured the knockout efficiency and found that the USP26 protein was completely depleted in Usp26 −/ Y testes (Fig 2B), indicating that the knockout mice were Usp26-null. The Usp26 −/ Y mice were viable and reached adulthood without any observable defects. We observed age-related phenotypes for fertility in Usp26 −/ Y mice: Both the pregnancy rate and litter size of Usp26 −/ Y mice were significantly reduced with increasing age (Fig 2C and D). It was highly conspicuous that 2-month-old Usp26 −/ Y mice did not produce XXY mice, but approximately 20% of the male mice sired by 6-month-old Usp26 −/ Y fathe