O-067 Biallelic variants in DLGAP5 cause spindle assembly defects and human early embryonic arrest

Abstract Study question What effects do DLGAP5 defects have on human early embryo development? Summary answer DLGAP5 deficiency disrupts normal spindle assembly through its interaction with TACC3, resulting in female infertility characterized as recurrent early embryonic arrest (REEA). What is known already REEA is a major factor contributing to failures in assisted reproductive technology. While genetic factors are essential, known gene variants explain only a small proportion of affected individuals, leaving many underlying genetic factors yet to be fully understood. The connection between spindle assembly and early embryonic development has become a prominent area of research, however, our comprehension of bipolar spindles in human oocytes and early embryos remains limited, indicating a need for further investigation into the key molecular players involved. Study design, size, duration In total, 488 female patients with infertility characterized as REEA were recruited from university-affiliated centers from November 2021 to December 2023. Participants/materials, setting, methods Whole exome sequencing was conducted on the REEA cohort to screen candidate variants. HeLa cells and Immunoprecipitation-mass spectrometry (IP-MS) were utilized to assess protein abundance and localization, as well as to identify altered interacting molecules associated with the candidate variants. Mutant mRNAs were expressed at the zygote stage to monitor subsequent embryonic development. A site-directed mutant mouse model was developed to investigate the pathogenic mechanisms in vivo, validated with patient oocytes and arrested embryos. Main results and the role of chance The study identified two nonsense variants, one frameshift variant, and one missense pathogenic variant in the DLGAP5 gene through whole-exome sequencing of three independent families from the REEA cohort. All affected individuals displayed a Mendelian recessive inheritance pattern. These variants significantly altered protein length, abundance, or localization, resulting in spindle abnormalities in HeLa cells and mouse zygotes. Furthermore, the injection of exogenous mutant DLGAP5 mRNA into mouse zygote and the construction of Dlgap5 site-directed mutant mice successfully replicated the patient phenotypes. Functional studies, both in vivo and in vitro, revealed that DLGAP5 deficiency disrupts normal spindle assembly through its interaction with TACC3. Limitations, reasons for caution This study was unable to observe the dynamic changes in spindle assembly in oocytes from patients with DLGAP5 variants due to ethical restrictions. Additionally, a larger patient cohort is needed, particularly multi-center and multi-ethnic studies, to further establish the relationship between DLGAP5 variants and female infertility. Wider implications of the findings These findings suggest that DLGAP5 is essential for spindle assembly in oocytes through its interaction with TACC3. This could position DLGAP5 as a novel molecular diagnostic marker and a potential target for interventions in female infertility related to REEA. Trial registration number No