Pathogenic variants in DLGAP5 cause female infertility characterized by oocyte maturation arrest and embryonic arrest

STUDY QUESTION: Can new genetic factors responsible for oocyte defects be identified in infertile women, especially for those with spindle assembly defects? SUMMARY ANSWER: We identified homozygous and compound heterozygous variants of DLGAP5 in three infertile individuals from two independent families. WHAT IS KNOWN ALREADY: Some genes have been found to be responsible for female infertility with oocyte maturation defects. During mitosis, DLGAP5 is involved in promoting microtubule polymerization and spindle formation. STUDY DESIGN, SIZE, DURATION: The DLGAP5 variants were identified by whole-exome sequencing in a cohort of 3627 female infertility patients diagnosed with oocyte maturation defects or embryonic development problems, and all participants were recruited from 2015 to 2023. Thirty-six hours after cell transfection, the expression levels of wild-type (WT) and mutant DLGAP5 were evaluated by western blot (n = 3 biological replicates). Human germinal vesicle (GV) oocytes retrieved from assisted reproductive procedure were introduced for cRNA (n = 3-5 oocytes per group) and antibody injection (n = 10-15 oocytes per group). Knock-in (KI) mouse model was generated by CRISPR-Cas9 and genotyping was performed at postnatal Days 10-15. Sexually mature females (6-10 weeks old) were used for fertility test (n = 6 mice per group, lasts 6-8 months), western blot (n = 3 biological replicates), IVF (n = 3 biological replicates), embryos collection (n = 3 biological replicates), immunofluorescence (n = 3 biological replicates), RNA-sequencing (RNA-seq, n = 3 biological replicates), and other functional assays between 2019 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: The DLGAP5 variants were identified by whole-exome sequencing and further confirmed by Sanger sequencing. Western blot was used to detect the expression of mutant DLGAP5 in HEK-293T cells after transfection. cRNA injection and immunofluorescence were performed to view the location of DLGAP5 in human oocytes. Knockdown of DLGAP5 by Trim-Away in human oocytes was conducted to observe the effect of DLGAP5 on spindle assembly and oocyte maturation. Then, Dlgap5 KI mice were constructed to mimic the phenotype of the affected individuals. After phenotypic assessment, western blot, IVF, assessment of embryonic development, chromosome counting, RNA-seq, and quantitative real-time PCR were performed to elucidate the pathological mechanism of DLGAP5 variants. MAIN RESULTS AND THE ROLE OF CHANCE: We identified homozygous nonsense DLGAP5 variant (NM_014750.5, c.431delA (p.Lys144Argfs*55)) in two affected sisters from family 1 and compound heterozygous variants (c.C847G (p.Pro283Ala) and c. C1202G (p.Thr401Ser)) in one infertile individual in family 2. p. Lys144Argfs*55 led to protein degradation (P < 0.0001) and p. Pro283Ala resulted in a significant decrease in protein level (P = 0.0021). DLGAP5 was located on the spindle and mutant did not alter its location. Knockdown of DLGAP5 in human oocytes impaired spindle assembly and led to oocyte maturation arrest (P = 0.0055). Homozygous (HO) Dlgap5 KI female mice showed reduced fertility (P < 0.0001) due to embryonic arrest at the 4-cell stage (P < 0.0001) and a low blastocyst formation rate (P < 0.0001). RNA-seq data of 4-cell embryos showed that differentially expressed genes (DEGs) were involved in cell cycle regulation, checkpoint control, and TGF-β signaling pathways, which may account for the embryonic development arrest. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to phenotypic differences between different DLGAP5 variants, more cases are needed to expand our understanding of the function of DLGAP5 in female infertility. Although RNA-seq data have shown the potential impact of DLGAP5 on cell cycle, embryonic development, and regulation of TGF-β signaling, more experiments are still requested to demonstrate the more direct role of DLGAP5 in oocyte and embryonic development. WIDER IMPLICATIONS OF THE FINDINGS: Our findings elucidated the role of DLGAP5 in human oocyte spindle assembly and the effects of the corresponding variants in the pathogenesis of oocyte maturation arrest, and these findings suggest that DLGAP5 is a novel maker for genetic counseling. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2024YFC2706600), the National Natural Science Foundation of China (82325021, 82288102, 32130029, 82371662, 82171643, 82201767, 82422033), the National Key Research and Development Program of China (2022YFC2702300), the New Cornerstone Science Foundation through the XPLORER PRIZE, L.W. is a SANS Exploration Scholar, and the Fund of Fudan University and Cao'ejiang Basic Research (24FCB01). None of the authors declare any conflict of interest. TRIAL REGISTRATION NUMBER: N/A.