ARTICLE https://doi.org/10.1038/s41467-018-08182-x OPEN Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella Ying Shen1, Feng Zhang 2,3,4, Fuping Li5,6, Xiaohui Jiang5,6, Yihong Yang7, Xiaoliang Li1, Weiyu Li2, Xiang Wang1, Juan Cheng8, Mohan Liu9, Xueguang Zhang1, Guiping Yuan10, Xue Pei9, Kailai Cai11, Fengyun Hu12, Jianfeng Sun13, Lanzhen Yan14, Li Tang1, Chuan Jiang1, Wenling Tu9, Jinyan Xu5,6, Haojuan Wu8, Weiqi Kong1, Shuying Li1, Ke Wang1, Kai Sheng1, Xudong Zhao14,15, Huanxun Yue5,6, Xiaoyu Yang16 & Wenming Xu1 1234567890():,; Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through sta- bilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation. 1 Department of Obstetrics/Gynecology, Joint Laboratory of Reproductive Medicine (SCU-CUHK), Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, 610041 Chengdu, China. 2 Obstetrics and Gynecology Hospital, Institute of Metabolism and Integrative Biology, School of Life Sciences, Fudan University, 200011 Shanghai, China. 3 State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, 211116 Nanjing, China. 4 Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, 200011 Shanghai, China. 5 Human Sperm Bank, West China Second University Hospital, Sichuan University, 610041 Chengdu, China. 6 Key Laboratory of Birth Defects and Related Disease of Women and Children (Sichuan University), Ministry of Education, 610041 Chengdu, China. 7 Center of Reproductive Medicine, West China Second University Hospital, Sichuan University, 610041 Chengdu, China. 8 Southwest Medical University, 646000 Luzhou, China. 9 Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center, 610041 Chengdu, China. 10 Analytical & Testing Center, Sichuan University, 610041 Chengdu, China. 11 Chengdu Research Base of Giant Panda Breeding, 610041 Chengdu, China. 12 Auckland University, 1142 Auckland, New Zealand. 13 Teaching Hospital of Chengdu University of TCM, 610041 Chengdu, China. 14 Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, 650000 Kunming, China. 15 Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, 650223 Kunming, China. 16 State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, The First Hospital, Nanjing Medical University, 210029 Nanjing, China. Correspondence and requests for materials should be addressed to X.Z. (email: [email protected]) or to H.Y. (email: [email protected]) or to X.Y. (email: [email protected]) or to W.X. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:433 | https://doi.org/10.1038/s41467-018-08182-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-018-08182-x perm defects are the direct causes of male infertility, which further identified the MMAF phenotypes of these two cases. We includes decreased sperm count, reduced sperm motility observed some spermatozoa with short tails, some with thick S 1,2 and abnormal morphology . Sperm motility is a critical short tails, and some with coiled tails, and some spermatozoa factor for normal fertilization, and over 80% of male infertility is appeared to have only the head region (Fig. 1c). In addition, a caused by impaired sperm motility3. Sperm morphology plays an variety of ultrastructural defects were detected in the sperm fla- essential role in sperm locomotion, and defective sperm mor- gella under transmission electron microscopy (TEM) (Fig. 1d). phology makes an assignable contribution to male infertility, For patient A (PA) integrated and regularly arranged OD and which consists of a wide range of phenotypes involving the head, ODF were observed, whereas the CP were missed in the mid- neck, mid-piece or tail of the spermatozoa. Among these, multiple piece of most flagella. In the principal piece of most flagella, some morphological abnormalities of the sperm flagella (MMAF) is ODF and OD were absent, the remainders were disorganized, and characterized by a mosaic of morphological abnormalities of the the CP could not be observed as well. For patient B (PB) the flagellum including coiled, bent, irregular, short or/and absent atypical 6 + 0 composition of axonemal microtubules was shown flagella4–6. Normal sperm flagella contain a 9 + 2 axonemal in the flagellar mid-piece; additionally, a complete lack of CP and arrangement of nine peripheral microtubule doublets (OD) and the irregular arrangement of the OD and ODF were revealed in two central microtubules (CP), surrounded by outer dense fiber the principal piece. (ODF) and fibrous sheath (FS)7. Almost all MMAF cases are The previous studies have reported that the homozygous loss- accompanied by visible ultrastructural derangement and/or of-function mutations of several genes could cause MMAF and defects, such as incomplete or disorganized axoneme arrange- have suggested that MMAF is a disorder with an autosomal- ment or absence of ODF/FS5,6,8. Any defects in these structures recessive inheritance pattern5,8,16,19. According to this, we would result in aberrant sperm morphology and further lead to performed WES on the two males to elucidate the underlying reduced motility or even sperm immobility. genetic causes of the MMAF phenotypes, and many homozygous Research is ongoing to investigate the molecular mechanisms variants were found in the two patients (Supplementary Data 1). of MMAF. Using knock-out (KO) mouse models, several genes Briefly, variants were removed if (1) the minor allele frequency have been found to be associated with sperm flagellar develop- was ≥1% in any database, including the gnomAD, ExAC Browser ment in mice9. The complete ablation of Spata6 caused impaired and 1000 Genomes Project, because the pathogenic variants development of the connecting piece and tight head-tail con- responsible for MMAF are rare in human populations; (2) the junction10. Subtle disorganization of the ultrastructure was heterozygous variant, because MMAF has been assumed to be an observed in Tekt4-null mice11. The sperm flagella of Tssk4-absent autosomal-recessive inheritance; (3) the variant affected: 3′ or 5′ mice represented morphological defects at the midpiece-principal untranslated regions, noncoding exons, or intronic sequences piece junction12. Mice lacking Odf2 showed axoneme defects13. except canonical splice sites; and (4) the variant was not predicted Ropn1-deficient mice exhibited structural defects in the principal damaging by PolyPhen-2, SIFT and MutationTaster tools. piece of sperm flagellum14. A significant disorganization in the Subsequently, the selected genes harboring candidate pathogenic fibrous sheath, as well as abnormal configuration of doublet variants from WES were evaluated by Human Protein Atlas microtubules, was observed in Cabyr-knockout male mice15. (http://www.proteinatlas.org), which contains tissue-specific pro- Although MMAF is not a rare autosomal-recessive inherited tein expression data, and the genes which are primarily or disorder in humans, the genetic etiology of a large proportion of specifically expressed in the testis were included in our research. MMAF cases remains elusive4–6. To date, mutations in only six Remarkably, after exclusion of frequent variants and application genes have been determined in humans related to MMAF: of technical and biological filters, two homozygous nonsense AKAP4, CCDC39, DNAH1, CFAP43, CFAP44, and CFAP69 mutations were identified in QRICH2 (Table 1): a homozygous (refs. 4,5,8,16–19); however, these could explain the mechanism of stop-gain mutation in exon 3 (c.192G>A [p. L64*]) in the only a few MMAF cases. Therefore, it remains necessary to individual PA and a homozygous stop-gain variant in exon 4 in improve the discriminative power of a single candidate gene in the individual PB (c.3037C>T [p.R1013*]). To clarify the putative human MMAF. contribution of these mutations to the MMAF phenotypes, we In this study, two homozygous nonsense mutations of the investigated the QRICH2 gene by Sanger sequencing in PA and glutamine rich 2 (QRICH2) gene are identified in two MMAF another affected individual (IV:3 and IV:5), their healthy parents patients from two consanguineous families through whole-exome (III:3 and III:4),